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Background: Candida albicans is one of the normal flora of the skin, mucous membranes and gastrointestinal tract. Vaginal candidiasis remains a significant problem in women of childbearing age, majority of the cases are caused by C. albicans and recurrence is common in spite of topical treatment. Objective: The purpose of this study is to develop the antifungal agent from the medicinal herbs traditionally used in Korea. Methods: In this study, extracts from roots of Paeonia japonica were examined for antifungal activities against C. albicans. Dried roots of Paeonia japonica were extracted with dichloromethane, methanol, water respectively and serially. Liquid column chromatography and thin layer chromatography were used to separate the fractions with antifungal activity, and mass spectrometric analysis was done to determine the mass. Results: Dichloromethane extract showed the highest antifungal activity aginst C. albicans. Result of fractionation and mass spectrometric analysis revealed that there were six materials: propanal, cytidine, hexadecanoic acid, cholesterol, octadecanoic acid and a unidentified material. Conclusion: Dichloromethane extract from Paeonia japonica could be a candidate for a new antifungal agent against C. albicans.