Candida albicans is the leading cause of vulvovaginal yeast infections; however, other species are becoming relevant in this niche. The spatial distribution of these fungi in the female genital tract remains poorly understood. In this study, swab samples were collected from 33 patients, first from the anterior vulva and then from the upper third and right lateral wall of the vagina: 16 were with symptoms of vulvovaginal candidiasis and 17 were without characteristic symptoms; furthermore, the genus and species of each isolate were identified. In vitro susceptibility testing for fluconazole and clotrimazole was performed for all isolates. Candida albicans was the most common species (63.6%), followed by Rhodotorula spp. (51.5%), and then Candida parapsilosis (15.2%). Rhodotorula spp. and C. parapsilosis were more commonly associated with colonization, and C. albicans with infection. Rhodotorula spp. isolates presented a low susceptibility to fluconazole, with the MIC ranging from 32 to >64 µg/mL. Differences in susceptibility to fluconazole and clotrimazole between the pairs of vaginal and vulvar isolates were found for Candida albicans, Rhodotorula spp., and Nakaseomyces glabratus. The results suggest that different niches may impact the susceptibility profiles of the isolates, as well as their different clinical behaviors.