Dried roots of Paeonia japonica were extracted with dichloromethane, methanol and water aerially. Silica gel column chromatography and thin layer chromatography were used to separate the fractions with antimicrobial activities, and mass spectrometry was used to determine the mass. Dichloromethane extract showed the highest antimicrobial activity. Dichloromethane extract from Paeonia japonica could be a candidate for a new antimicrobial agent against MRSA.