Objective To research the anti-fungal spectrum and activity of the cream containing 1% naftifine-0.25% ketoconazole compared with other two creams that contain of 2% ketoconazole and of 1% terbinafine, respectively. Methods The agar diffusion method was used to judge drug sensitivity. Twenty-nine isolates of pathogenic fungi belonging to 11 species from clinic and three species of Malassezia standard stains were enrolled into the experiment. Organism suspension of each species was spread on the surface of the plate of the optimal media containing 2% agar. Then wells were made in the plate and three types of cream were put in each well respectively. After seven-day incubation, the diameter of the inhibition zone around the well full of each cream was observed and recorded. Results The inhibition zone around the well full of 1% naftifine-0.25% ketoconazole cream for all experiment isolates (Dermatophytes, Candida spp., Sporothrix schenkii, Fonsecaea pedrosoi, Fusarium graminearum, Malassezia furfur, M. globosa and M. sympodialis) was observed, with the mean diameter of 45.46mm. Similarly, the mean diameter of inhibition zone of 2% ketoconazole cream for all experiment isolates was 23.92mm. About 1% terbinafine cream, the mean diameter was 29.81mm but there was no inhibition zone observed around Candida krusei and Candida albicans mycelial-form. There were significant significances for mean diameters of the inhibition zone when comparing 1% naftifine-0.25% ketoconazole cream with 2% ketoconazole cream (P=0.000) and with 1% terbinafine cream (P=0.000). Conclusion The anti-fungal spectrum of 1% naftifine-0.25% ketoconazole cream is wider than that of 1% terbinafine cream. The antifungal activity of 1% naftifine-0.25% ketoconazole cream is ber than that of 2% ketoconazole cream and 1% terbinafine cream.