ABSTRACTTo investigate the combined function of the novel oral mTOR inhibitor, everolimus, with antifungal agents and their potential mechanisms againstExophiala dermatitidis, the CLSI microliquid-based dilution method M38-A2, chequerboard technique, and disk diffusion testing were performed. The efficacy of everolimus was evaluated in combination with itraconazole, voriconazole, posaconazole, and amphotericin B against 16 clinically isolated strains ofE. dermatitidis. The synergistic effect was determined by measuring the MIC and fractional inhibitory concentration index. Dihydrorhodamine 123 was used for the quantification of ROS levels. The differences in the expression of antifungal susceptibility-associated genes were analyzed following different types of treatment.Galleria mellonellawas used as thein vivomodel. While everolimus alone showed minimal antifungal effects, combinations with itraconazole, voriconazole, posaconazole, or amphotericin B resulted in synergy in 13/16 (81.25%), 2/16 (12.5%), 14/16 (87.75%), and 5/16 (31.25%) of isolates, respectively. The disk diffusion assay revealed that the combination of everolimus and antifungal drugs showed no significant increase in the inhibition zones compared with the single agent, but no antagonistic effects were observed. Combination of everolimus and antifungal agents resulted in increased ROS activity (everolimus + posaconazole versus posaconazole [P< 0.05], everolimus + amphotericin B versus amphotericin B [P< 0.002]). Simultaneously, compared to mono-treatment, the combination of everolimus + itraconazole suppressed the expression ofMDR2(P< 0.05) and the combination of everolimus + amphotericin B suppressed the expression ofMDR3(P< 0.05) andCDR1B(P< 0.02).In vivo, combinations of everolimus and antifungal agents improved survival rates, particularly the combination of everolimus + amphotericin B (P< 0.05). In summary, thein vivoandin vitroexperiments performed in our study suggest that the combination of everolimus with azoles or amphotericin B can have synergistic effects againstE. dermatitidis, potentially due to the induction of ROS activity and inhibition of efflux pumps, providing a promising new approach for the treatment ofE. dermatitidisinfections.IMPORTANCECancer patients withE. dermatitidisinfection have high mortality if untreated. Clinically, the conventional treatment ofE. dermatitidisis poor due to the long-term use of antifungal drugs. In this study, we have for the first time investigated the interaction and action mechanism of everolimus combined with itraconazole, voriconazole, posaconazole, and amphotericin B onE. dermatitidis in vitroandin vivo, which provided new ideas and direction for further exploring the mechanism of drug combination and clinical treatment ofE. dermatitidis.