The effects of five antifungal drugs, fluconazole, itraconazole, micafungin, miconazole, and voriconazole, on cytochrome P450 (CYP) 2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4′-hydroxylation, and CYP3A4-mediated nifedipine oxidation activities in human liver microsomes were compared. In addition, the effects of preincubation were estimated to investigate the mechanism-based inhibition. The IC₅₀ value against tolbutamide hydroxylation was the lowest for miconazole (2.0 μM), followed by voriconazole (8.4 μM) and fluconazole (30.3 μM). Similarly, the IC₅₀ value against S-mephenytoin 4′-hydroxylation was the lowest for miconazole (0.33 μM), followed by voriconazole (8.7 μM) and fluconazole (12.3 μM). On the other hand, micafungin at a concentration of 10 or 25 μM neither inhibited nor stimulated tolbutamide hydroxylation and S-mephenytoin 4′-hydroxylation, and the IC₅₀ values for itraconazole against these were greater than 10 μM. These results suggest that miconazole is the strongest inhibitor of CYP2C9 and CYP2C19, followed by voriconazole and fluconazole, whereas micafungin would not cause clinically significant interactions with other drugs that are metabolized by CYP2C9 or CYP2C19 via the inhibition of metabolism. The IC₅₀ value of voriconazole against nifedipine oxidation was comparable with that of fluconazole and micafungin and higher than that of itraconazole and miconazole. The stimulation of the inhibition of CYP2C9-, CYP2C19-, or CYP3A4-mediated reactions by 15-min preincubation was not observed for any of the antifungal drugs, suggesting that these drugs are not mechanism-based inhibitors.