Antifungal agents can effectively treat mucosal candidiasis; however, their use can lead to colonization with less susceptible species and to resistance among normally susceptible strains. Oral and vaginal Candida isolates obtained at 3 points over 2 years from human immunodeficiency virus (HIV)-seropositive and at-risk HIV-seronegative women were identified by species and were evaluated for in vitro fluconazole susceptibility. Prevalence of non-C. albicans strains increased over time, and these strains were more likely among women reporting current antifungal use. Among C. albicans isolates, resistance was rare, with no evidence for progressive reduction in susceptibility over time. Among non-C. albicans isolates, reduced susceptibility occurred frequently and increased with time. HIV-seropositive women were more likely to have non-C. albicans isolates with reduced susceptibility as were women reporting current antifungal use. This evolution and section of mucosa-colonizing Candida species with reduced susceptibility could play a critical early role in the development of antifungal resistance among C. albicans isolates responsible for refractory candidiasis.