Unmasking the Fungicidal Potency and Multifaceted Mechanisms of Nutmeg Essential Oil Against Candida auris

<b>Background</b>: <i>Candida auris</i> has emerged as a multidrug-resistant fungal pathogen, presenting significant clinical challenges worldwide. Although considerable progress has been made in antifungal research, the specific mechanisms underlying drug resistance in <i>C. auris</i> remain incompletely understood. To overcome this problem, natural compounds can be used as valuable alternatives. The present study aimed to evaluate the antifungal activity of NEO against <i>C. auris</i> and to understand the functional mechanisms underlying its antifungal activity. <b>Methods</b>: The antifungal activity of NEO against <i>C. auris</i> strain CBS10913T was examined using broth microdilution and spot assays to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Mechanistic investigations were performed using phenotypic-, biochemical-, and fluorescence-based assays to evaluate its effects on cell wall integrity, membrane permeability, efflux pump activity, oxidative stress, lipid peroxidation, biofilm formation, and host cell adherence. Hemolytic assays were performed to evaluate preliminary biocompatibility. <b>Results</b>: During our study, we found that NEO showed strong fungicidal activity against <i>C. auris,</i> with an MIC of 500 µg/mL and an MFC of 650 µg/mL, and disrupted fungal cell wall integrity, significantly reduced ergosterol content, and inhibited efflux pump activity, leading to increased accumulation of fluorescent substrates. NEO induced increased intracellular reactive oxygen species, leading to oxidative-mediated lipid peroxidation and DNA damage. Moreover, NEO also suppressed stress biofilm formation, reduced metabolic activity, and decreased adherence to buccal epithelial cells, and it showed negligible hemolytic activity up to 2× MIC, indicating preliminary biocompatibility. <b>Conclusions</b>: This study demonstrates that NEO utilizes broad antifungal activity through multiple functional and phenotypic mechanisms, including disruption of membrane integrity, inhibition of efflux pump, induction of oxidative stress, and suppression of biofilm formation. Although the direct effects on pathogenicity-related genes or proteins were not studied, the findings still show NEO as a promising natural antifungal agent.