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Current treatment options for vulvovaginal candidiasis due to Candida albicans include over-the-counter and prescription antifungal agents. Fluconazole has been used extensively with an unknown impact on susceptibility. To investigate antifungal susceptibility trends in clinical vaginal isolates of C. albicans from 1986 to 2008, microdilution susceptibility was performed on randomly selected single isolates. Minimum inhibitory concentrations (MICs) were determined for: fluconazole, clotrimazole, miconazole, ketoconazole, itraconazole, voriconazole, flucytosine and amphotericin B. The MIC90 for each drug was then calculated for the time periods: 1986–1989, 1992–1996 and 2005–2007. A total of 250 C. albicans vaginal isolates were included. The MIC90 (mcg ml−1) for fluconazole was 0.25, 0.5 and 0.5 mcg ml−1 for each grouping, respectively. The corresponding MIC90 for flucytosine was 1, 2 and 8 mcg ml−1, respectively. The MIC90 for the remaining agents remained unchanged across time periods mentioned. Of note, the percentage of isolates with MIC ≥1 and ≥2 mcg ml−1 for fluconazole increased from 3% to 9% over the study period. Although the C. albicans MIC90 to fluconazole in vaginal isolates has not shown a clinically significant increase since 1986, there is an increasing number of isolates with elevated MICs. The implications of this increase are unknown, but given the achievable vaginal concentrations of fluconazole, reduced susceptibility may have clinical relevance.