The effect of fluconazole on the susceptibility of Candida isolates recovered from women infected with human immunodeficiency virus (HIV) was evaluated in a randomized, double-blind, placebo-controlled trial. Women with CD4+ cell counts of ≤ 300 cells/mm3 received either fluconazole (200 mg/week) or placebo as prophylaxis. The antifungal susceptibility of specimens was evaluated. One patient who received fluconazole and 2 patients assigned to placebo had Candida albicans isolates recovered that were resistant to fluconazole (MIC, ≥ 64 μg/mL). Eleven patients assigned fluconazole and 4 patients assigned placebo had non-albicans Candida strains (all Candida glabrata) recovered that were resistant to fluconazole. There was significant azole cross-resistance among the non-albicans Candida species isolates. Although the rate of azole resistance did not significantly increase after fluconazole prophylaxis, there was a trend toward more in vitro azole resistance in C. glabrata isolates from patients assigned fluconazole. Moreover, the majority of resistant vaginal isolates of Candida species were recovered after initiation of open-label fluconazole use.