Kaempferia rotunda (Zingiberaceae), known as kunci pepet or kunir putih in Indonesia, has been traditionally used in abdominal pain, sputum laxative, wounds and diarrhea colic disorder. This study was conducted to isolate and to investigate antimutagenic activity of some flavanones from K. rotunda.The milled dried rhizoma of K. rotunda (3 kg) was extracted exhaustively with methanol. The methanol extract was partitionated three times by n-hexane, chloroform, and ethyl acetate respectively. Each fraction was fractionated by vacuum liquid chromatography (VLC) and purified by column chromatography gravitation. Identification structures of all pure compounds were elucidated based on spectroscopic methods (UV, IR, and NMR) and compared to the spectroscopic previously reported data. Antimutagenic activity test was observed in vivo based on the number of micronucleated polychromatic cell erythrocytes (MNPCE) from male Balb-c mice (8–12 week) induced by cyclophosphamide.From the dried and milled rhizoma of K. rotunda, three known flavanones, namely 5-hydroxy-7-methoxyflavanone (1), 7-hydroxy-5-methoxyflavanone (2), and 5,7-dihydroxyflavanone (3) were isolated. The methanol extract and isolated flavanones from K. rotunda showed significant antimutagenic effect compared to control group.