Candida species are increasingly common fungal pathogens that pose a significant threat to immunocompromised individuals, particularly in the context of increasing resistance to currently available antifungals. In 2022, the World Health Organization (WHO) listed five Candida species among the critical and high-priority groups of fungal pathogens due to their high prevalence and rising drug resistance: Candida albicans, Candidozyma auris (syn. Candida auris), Nakaseomyces glabratus (syn. Candida glabrata), Candida parapsilosis, and Candida tropicalis (World Health Organization, 2022). With only four chemical classes of antifungals in clinical use—azoles, polyenes, echinocandins, and pyrimidine analogues—the growing incidence of drug-resistant Candida strains presents a therapeutic deadlock (Arastehfar et al., 2020). This situation severely limits treatment effectiveness, making candidiasis harder to manage and posing the threat of a silent pandemic. In light of this growing challenge, there is a pressing need to explore new therapeutic approaches, including the use of alternative antifungal agents (Kriegl et al., 2025), combination therapies (Soulaimani et al., 2021), and compounds targeting virulence rather than growth (Cui et al., 2015). By inhibiting virulence traits such as adhesion, production of hydrolytic enzymes, tissue invasion, and biofilm formation, candidate anti-virulence compounds could disarm the pathogen without necessarily killing it. This approach may reduce the selective pressure for resistance development, preserve the host’s microbiota, and minimize adverse side effects (Buroni and Chiarelli, 2020).

Recently, there has been a growing interest in discovering new and safe antimicrobial molecules derived from natural sources, particularly from aromatic and medicinal plants (Nazzaro et al., 2017). Essential oils (EOs) are complex mixtures of volatile plant-derived compounds that have demonstrated promising antifungal activity against a wide range of fungal pathogens, including Candida species (Kalemba and Kunicka, 2003; Tabassum and Vidyasagar, 2013; Nazzaro et al., 2017; Saracino et al., 2022). In this context, EOs derived from thyme, cinnamon and clove have attracted particular attention due to their strong activity against Candida species (Pina-Vaz et al., 2004; Pinto et al., 2009; Gucwa et al., 2018; Tran et al., 2020). Thyme EO is mainly characterized by the presence of thymol, cinnamon EO by cinnamaldehyde, and clove EO by eugenol (Yu, 2025). These phenolic compounds are known to exert potent antifungal effects, primarily through disruption of fungal cell membrane integrity, which may ultimately lead to fungal cell death (Ahmad et al., 2011; Shreaz et al., 2013; Niu et al., 2020; Didehdar et al., 2022).

Despite the broad antimicrobial potential of EOs, their inhibitory effects against clinically relevant antifungal-resistant strains, as well as their potential impact on Candida virulence factors, remain poorly understood.

Germ tube formation and the secretion of proteases are two major virulence factors in Candida species. Notably, the germ tube is a typical morphological feature of C. albicans, serving as a reliable marker for its identification. It consists of a small initial filamentous outgrowth from the yeast cell, indicating the start of hyphal formation. This morphological shift from yeast to hyphal form is crucial to the virulence of C. albicans (Mayer et al., 2013). Hyphal formation is associated with several pathogenic traits, including adherence to epithelial and endothelial surfaces, host tissue invasion, iron acquisition, escape from phagocytes, and immune evasion (Jacobsen et al., 2012). Another key virulence factor in Candida species is secreted proteases. These enzymes can break down a wide range of host proteins and are considered among the most critical contributors to pathogenicity. Proteases facilitate adhesion, promote tissue invasion, and contribute to host cell damage (Silva et al., 2014). Although the function of secreted proteases has been well characterized in C. albicans, their roles in non-albicans species remain underexplored.

In this study, we analyzed the composition of three commercial EOs—cinnamon, thyme and clove—and tested their inhibitory activity in comparison to established azole and echinocandin antifungals on a panel of eighteen Candida strains covering nine different species and including drug-resistant isolates. Furthermore, the impact of the two most effective EOs on key Candida virulence factors was investigated, focusing on germ tube formation in Candida albicans and protease production in non-albicans Candida species. Together, these approaches allowed us to assess not only the antifungal potential of essential oils but also their impact on key virulent traits, providing new insights into their possible role as alternative or complementary treatments against Candida infections.

The fight against fungal infections is becoming more challenging as the armamentarium of available antifungals remains limited and resistance continues to increase. This scenario poses a pressing need to develop new antifungal compounds or alternative therapeutic approaches. In recent years, EOs have attracted interest due to their long history of use for their antimicrobial properties in treating viral, bacterial, and fungal infections (Reichling et al., 2009). Additionally, they have been investigated for use in topical creams (Nejati et al., 2015), as food preservatives (Tongnuanchan and Benjakul, 2014), and as biocides in sustainable agriculture (Assadpour et al., 2024). The present study focused on investigating the antifungal and anti-virulence potential of three EOs against a panel of eighteen diverse Candida strains, including reference, clinical, and multidrug-resistant strains from nine different species.

GC-MS analysis revealed that the major components of cinnamon, thyme, and clove EOs were cinnamaldehyde (72.43%), thymol (74.6%), and eugenol (81.74%), respectively. They belong to two major classes of bioactive molecules—phenylpropanoids (cinnamaldehyde and eugenol) and monoterpenes (thymol). Their high relative abundance indicates that these compounds likely play a central role in the biological activity of each oil. Previous GC-MS studies have shown that cinnamon and clove EOs may consist of a limited number of components. For instance, cinnamon EO was reported to contain only five identified constituents, with cinnamaldehyde as the major compound (96.80%) (Keshvari et al., 2013), while clove EO exhibited a similarly simple profile, with four main compounds dominated by eugenol (74.34%) (Trifan et al., 2021). In the case of cinnamon EO, the predominance of cinnamaldehyde aligns with previous findings in Cinnamomum zeylanicum, as reported by (Saleem et al., 2015; Shahina et al., 2018; Tran et al., 2020), where cinnamaldehyde was identified as the major compound. In the case of thyme EO, the high content of thymol is consistent with earlier studies in Thymus vulgaris (Hudaib et al., 2002; Borugă et al., 2014; Baj et al., 2020). Similarly, the clove EO analyzed in this study showed a dominant presence of eugenol, which is consistent with the characteristic chemical profile of Syzygium aromaticum described in previous works (Didehdar et al., 2022; Kiki, 2023; Momo et al., 2024). Overall, these findings confirm the consistency of our results with previously reported chemical profiles of the considered EOs while also highlighting slight differences that can be associated with factors such as geographic origin, plant chemotype, extraction conditions, and seasonal variation.

In this study, MIC values for cinnamon, thyme and clove EOs were expressed in % (v/v), as this unit remains the most widely used in the literature. The results revealed distinct inhibition responses to EOs, with cinnamon EO showing the strongest effect, followed by thyme and clove EOs. The strong antifungal activity of cinnamon EO is mainly attributed to cinnamaldehyde, which acts by disturbing the ergosterol biosynthesis pathway and directly binding to ergosterol within the fungal membrane, thus weakening membrane integrity (da Nóbrega Alves et al., 2020; Khan et al., 2013). In addition, cinnamaldehyde and related compounds strongly inhibit plasma membrane ATPase activity, which interferes with ATP-dependent efflux processes (Shreaz et al., 2013; Shreaz et al., 2011). In addition, cinnamon EO can also induce spindle defects and G2/M cell cycle arrest in C. albicans, highlighting its potential to disrupt fungal growth through multiple cellular targets (Shahina et al., 2018). Similarly, thyme EO, rich in thymol, exhibited moderate to strong inhibitory effects, in agreement with earlier reports highlighting its ability to alter ergosterol and lipid organization, increasing permeability and causing cytoplasmic leakage (Ahmad et al., 2011; de Castro et al., 2015). While both compounds share membrane-disrupting activities, thymol additionally induces mitochondrial dysfunction, reactive oxygen species accumulation, and vacuolar/cytoplasmic disorganization, highlighting distinct effects on fungal cell structure (Braga and Ricci, 2011). Clove EO was generally less potent than cinnamon and thyme EOs; however, it still showed antifungal activity mainly attributed to eugenol, which disrupts the fungal membrane, causes ion and macromolecule leakage, and induces oxidative stress that weakens mitochondrial function and cellular homeostasis in Candida species (Didehdar et al., 2022).

Among the tested MDR strains, C. parapsilosis Cpar_CDC317_MDR showed the most pronounced sensitivity to EOs, with a fourfold decrease in MIC for CEO and a twofold decrease for TEO compared to its parental strain (Cpar_CDC317_WT). These reductions occurred despite the presence of mutations in FKS1 and MRR1, which confer echinocandin resistance by reducing β-1,3-glucan synthase binding and azole resistance via expression of efflux pumps, respectively (Nunez-Rodriguez et al. n.d.). Similarly, N. glabratus Ngla_CBS138_MDR exhibited a twofold decrease in MIC values for thyme and clove EOs relative to its parental strain (Ngla_CBS138_WT), even though it carries mutations in FKS1-3, ERG3, IZH3 and CSF1. These mutations contribute to echinocandin resistance through altered glucan synthase activity, and azole resistance via ergosterol biosynthesis changes, and affect cell wall integrity and zinc homeostasis (Ksiezopolska et al., 2021). In contrast, N. glabratus Ngla_CST34_MDR, which harbors mutations in FKS1, FKS2 and PDR1, along with a partial duplication in chromosome E, displayed MIC values similar to its parental strain (Ngla_CST34_WT) across all tested EOs. These alterations confer echinocandin resistance via changes in β-1,3-glucan synthase and azole resistance through increased efflux pump activity (Ksiezopolska et al., 2021). Overall, these findings suggest that EOs act through mechanisms distinct from those of conventional antifungal drugs and may overcome classical resistance mechanisms, highlighting the need for further investigation into their specific molecular targets and pathways.

Interestingly, C. auris, known for its global spread and emerging resistance, showed notable susceptibility to the tested EOs, with comparatively lower MICs than other Candida species. In particular, clades II, III and V were the most sensitive. In contrast, clades I and IV exhibited higher MICs, indicating reduced susceptibility. A recent comparative review reported considerable variation in antifungal resistance; clades I, III, and IV are characterized by multidrug resistance, particularly to fluconazole and amphotericin B, which complicates treatment, whereas clades II, V and VI show much lower resistance (da Silva et al., 2025). Moreover, the expression of virulence factors such as adhesion expression, biofilm formation and hydrolytic enzyme production are generally higher in the more outbreak-prone clades (I, III, and IV) compared to the less resistant clades (Kim et al., 2024; Santana et al., 2024).

C. albicans (both Calb_SC5314_WT and Calb_CL), C. tropicalis (Ctro_CSPO_WT), and C. dubliniensis (Cdub_CL), three closely related species, exhibited similar MICs for TEO, with C. dubliniensis being more susceptible to CEO. Comparative studies have shown that the three species often display similar susceptibilities to EOs, despite the presence of species-specific differences (Pozzatti et al., 2008; Pozzatti et al., 2010; Mandras et al., 2016). C. parapsilosis (Cpar_CDC317_WT) and its sibling species C. orthopsilosis (Cort_90-125_WT) followed the same trend, reinforcing the idea that phylogenetically related species tend to respond similarly to EOs. However, C. metapsilosis (Cmet_BP57_WT) displayed lower MIC values when exposed to CEO and TEO, indicating greater sensitivity and suggesting that even small genetic differences within the complex can lead to distinct responses. P. kudriavzevii (Pkud_CL) exhibited the highest MICs for both CEO and TEO. Although classified in the WHO fungal priority pathogens list as a medium-priority species, it demands further investigation for new antifungals. P. kudriavzevii is intrinsically resistant to fluconazole, and recent reports have documented emerging resistance to other antifungal agents as well (Badiee et al., 2017; Jamiu et al., 2021; Zhang et al., 2023). Taken together, these results reinforce the idea that CEO and TEO, rich in cinnamaldehyde and thymol, possess notable antifungal activity, particularly against strains less responsive to azoles. Their role as complementary agents in managing resistant Candida infections merits further exploration.

Both cinnamon and thyme EOs disrupted C. albicans filamentation but through different effects. Cinnamon EO mainly shortened and partially inhibited germ tubes, while thyme EO fully hindered true hyphae, a key virulence factor in C. albicans, and promoted pseudohyphae formation. These differences likely result from distinct mechanisms; however, the molecular basis of these contrasting effects is still unknown.

Cinnamaldehyde has been reported to decrease germ tube formation, along with adhesion and hydrolytic enzyme production, collectively weakening virulence factors of C. albicans (Pootong et al., 2017; El-Baz et al., 2021; Essid et al., 2019). Previous studies reported that cinnamon EO inhibited germ tube formation in C. albicans isolates, with inhibition percentages ranging from 44.7% to 82.9% at concentrations of 62.5-500 μg/mL (Carvalho et al., 2018). At subinhibitory concentrations (62.5 and 31.25 μg/mL), cinnamaldehyde significantly reduced germ tube formation in a dose-dependent manner (Pootong et al., 2017). Additionally, both cinnamon bark and clove EOs, containing cinnamaldehyde and eugenol, inhibited hyphal morphogenesis, leading to substantial reductions in germ tube formation (Shahina et al., 2022). In contrast, thyme EO has been shown to promote pseudohyphal structures in C. albicans; a study observing morphological changes reported that exposure to thyme (and tea tree) oils led to noticeable alterations in colony and cell morphology, particularly including pseudohyphae appearance (Rajkowska et al., 2014). Furthermore, Alshaikh et al. reported that thyme EO inhibited germ tube formation by over 80% in fluconazole-resistant strains at ½ MIC (Alshaikh and Perveen, 2021). Thymol was found to significantly reduce hyphal formation in a dose-dependent manner at sub-inhibitory concentrations: at 31.25 μg/mL, around 46% of cells formed germ tubes, while at 62.5 μg/mL, only 29% were able to produce them (Braga et al., 2007).

Another virulence factor in Candida species is the protease production, an important group of hydrolytic enzymes contributing to their pathogenicity. C. albicans possesses a well-characterized family of ten genes (secreted aspartyl proteases; SAP1-SAP10) with roles linked to yeast growth, hyphal invasion, and stress adaptation (Naglik et al., 2003). Non-albicans Candida species also produce secreted proteases, but with fewer members or different types (Tóth et al., 2019; Zaugg et al., 2001; D Gilfillan et al., 1998). For instance, C. dubliniensis, C. tropicalis, and C. parapsilosis encode SAP homologs with varying sequence similarity and expression profiles, while N. glabratus lacks classical SAPs and instead produces yapsins (YPS1-YPS11), which are glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases involved in cell wall remodeling and host interaction (Kaur et al., 2007; Pandey et al., 2018). In contrast, emerging species such as C. auris carry SAP gene homologues (Jenull et al., 2021; Kim et al., 2023; Chatterjee et al., 2015), though their exact functions in virulence remain under investigation. Interestingly, protease production in C. auris differs between clades, which was previously demonstrated between clades I and III, with the former showing greater virulence in infection models (Wang et al., 2018; Fan et al., 2021). To our knowledge, this is the first study comparing the five clades of C. auris in terms of protease activity and evaluating the effect of EOs on it.

Cinnamon and thyme EOs’ effects on protease activity revealed distinct patterns across the tested Candida species, with several strains showing marked reductions in protease production. For instance, Caur_I_WT and Ctro_CSPO_WT showed a strong decrease in their protease activity when exposed to cinnamon EO, while Caur_II_WT and Caur_III_WT displayed total inhibition when treated with thyme EO. In addition, Caur_IV_WT and Pkud_CL exhibited a significant reduction in their protease activity. Another noteworthy finding was observed in Ngla_CST34_MDR, where protease activity was notably affected by both oils compared to its parental strain. Even though high antifungal MICs were needed, inhibiting virulence factors in such strains is a valuable strategy because it can diminish their pathogenic potential. C. metapsilosis has often been reported as unable to produce detectable levels of proteases, a feature that may be influenced by strain differences or methodological factors (Neji et al., 2017; Gago et al., 2014; Sabino et al., 2011). Compared with C. parapsilosis sensu stricto, relatively few studies have focused on the enzymatic activities of C. orthospilosis and C. metapsilosis, leaving important gaps in our understanding of their virulence potential. Nevertheless, these species should not be underestimated, as they are increasingly identified as emerging pathogens capable of acquiring antifungal resistance and causing severe clinical infections (Del Olmo et al., 2025; Del Olmo and Gabaldón, 2024). In a few cases, such as C. tropicalis exposed to thyme EO or P. kudriavzevii exposed to cinnamon EO, we observed a slight increase in secreted protease activity at sub-inhibitory concentrations, which was demonstrated by the decrease of the Pz value in comparison to the control. This may indicate an adaptive stress that helps to control virulence in the presence of bioactive molecules. Our findings show evidence that exposure to subinhibitory concentrations of EOs can reduce and even suppress protease expression in Candida species. Most previous studies have focused on C. albicans (Pootong et al., 2017; Khan et al., 2014; Shreaz et al., 2012), while data on non-albicans Candida species remain limited. To our knowledge, this study provides the first comparative assessment of protease expression in response to EOs across multiple non-albicans Candida species.

Our study provides compelling evidence of potent antifungal activity of EOs against a broad range of Candida species and strains, extending earlier results and underscoring their potential as alternative or complementary therapeutic options. Furthermore, our results suggest that EOs can weaken pathogenicity even without the complete elimination of fungal cells, as evidenced by their ability to inhibit germ tube formation in C. albicans and reduce secreted protease activity in non-albicans species. Importantly, the strong activity of EOs across diverse isolates indicates a prospect not only for treating a broad range of fungal infections but also a potential to help control multidrug-resistant strains in healthcare settings. Nevertheless, further investigations are necessary to translate these promising in vitro results into clinical applications, including toxicity assessment in suitable animal models such as Galleria mellonella and the optimization of delivery systems through advanced formulations like nanoparticles to increase stability, bioavailability, and therapeutic efficacy.