Objective: To study the effect of fluconazole on ergosterol bios ynthesis in fungi by the method of thin-layer chromatography (TLC). Methods: Saponification and extraction of the nonsaponifiable lipids （NSLs） of fungi were carried out after 24h of incubation with varied concentrations of fluconazole. The components of the NSLs were separated by TLC and the concentr ations of ergosterol and lanosterol were determined. Results: Fluc onazole inhibited ergosterol biosynthesis and caused the concentration of lanost erol increased in a dose-dependent manner in fungi. When fluconazole inhibited ergosterol biosynthesis by 85%, the growth of fungi was also inhibited by about 85%. Conclusion: The effect of antimycotic agents on ergosterol biosynthesis in fungi can be studied by determining the concentrations of ergoster ol and lanosterol by TLC. The method can be used to study the activity and mecha nism of antimycotic agents because of its accuracy and easy operation.