9-Hydroxycrisamicin A, a new cytotoxic isochromanquinone antibiotic, was isolated from a soil microorganism SA246 which was identified as Micromonospora sp. The molecular formula of 9-hydroxycrisamicin A was determined as C32H22O13 based on the HRFAB-MS analysis, and the structure was determined by various NMR experiments. 9-Hydroxycrisamicin A showed weak antimicrobial activity against Gram-positive bacteria and strong cytotoxic activity against some human cancer cell lines such as SK-OV-3 (ovarian), HCT15 (colon), SK-MEL-2 (melanoma), A549 (lung), XF498 (central nervous system) with ED50 of 0.47-0.65 microgram/ml.