In vitro antifungal activities of polygodial were investigated against several fungal pathogens. Polygodial showed strong antifungal activity, comparable to amphotericin B, against yeast-like fungi Candida albicans, C. utilis, C. krusei, Cryptococcus neoformans, Saccharomyces cerevisiae and also filamentous fungi including Trichophyton mentagrophytes, T. ruburum, and Penicillium marneffei. Other strains such as Aspergillus fumigatus, A. flavus, P. chrysogenum, C. lipolytica, and C. tropicalis showed moderate to low susceptibility to polygodial. The anti-fungal activity of polygodial was generally not reduced by several susceptibility-testing conditions such as medium, incubation temperature, inoculum size, and medium pH. However, polygodial's antifungal activity was strongly increased at acidic conditions. Unlike amphotericin B, polygodial did not show any hemolytic activity and also its antifungal activity was not diminished in the presence of ergosterol. Based on killing kinetics against growing and nongrowing C. albicans, polygodial showed strong and rapid fungicidal activity.