Rhodotorula mucilaginosa was isolated from a patient with onychomycosis, and identification was confirmed by morphological and cultural characteristics as well as by DNA molecular analysis. Antifungal agents naftifine (10 mg/mL, active substance in Exoderil) and bifonazole (10 mg/mL, active substance in Canespor) were tested in different concentrations to assess in vitro effects on fungal growth and carotenoid synthesis. The antifungal mechanisms of action of naftifine and bifonazole against R. mucilaginosa isolates were similar and affected the biosynthetic pathway of ergosterol. For the first time, this research demonstrates that naftifine affects the carotenoid biosynthetic pathway, producing depigmentation of R. mucilaginosa in solid and liquid media. Furthermore, depigmentation was a reversible process; naftifine-treated yeast cells that were depigmented resumed carotenoid production upon transfer to fresh media. Raman and UV-vis spectrophotometry in conjunction with chromatographic analysis detected changes in carotenoids in yeast cells, with torulene decreasing and B-carotene increasing after repigmentation. Transmission electron micrographs revealed critical ultrastructural modifications in the depigmented cells after naftifine treatment, i.e., a low-electron-density cell wall without visible mucilage or lamellate structure.