There are a large number of bioactive sesquiterpene lactones from compositae plants including Hemistepita lyrata Bunge. In the present study, we purified two sesquiterpene lactones, Hemistepsin A and B, from H. lyrata and evaluated their antimicrobial activities against Malassezia obutusa. Chromatographic separation was used for the preparation of Hemistepsin A and B, and the identity of these compounds was confirmed by NMR. Strong inhibition of growth of M. obutusa was obtained with all doses of Hemistepsin A tested. Moreover, antifungal activity of Hemistepsin A occurred in a dose-dependent manner. Hemistepsin B also showed potent antifungal activity at the dose of $800{mu}m/disc$. From these results, it was suggested that Hemistepsin A and B be beneficial for the preparation of the useful agent for treating scalp diseases occurred by dandruff-causing Malassezia species.