An ATP bioluminescence assay as a rapid reference method for fluconazole (FLCZ) susceptibility testing of dermatophytes, as well as yeasts, was developed and evaluated by comparing it with viability, turbidity and fungal protein content-based conventional methods. FLCZ susceptibility results obtained with strains of Candida albicans and dermatophytes by the bioluminescence method in high-resolution medium were well correlated with those obtained by conventional methods currently used in clinical microbiology laboratories or reported previously, including a broth dilution method by the National Committee for Clinical Laboratory Standards (NCCLS). Thus, ATP bioluminescence assay can be used to monitor fungal growth in liquid culture media. The procedure has considerable potential for the rapid testing of FLCZ susceptibility of dermatophytes and other fungi.