Strain DS4, a soil microorganism producing an oxygen radical-generating antibacterial substance, bruceanol B, was isolated, and cultural conditions for the antibiotic production were optimized. Bruceanol B was separated by ethyl acetate extraction, silica gel and Sephadex LH-20 gel column chromatographies, and high-performance liquid chromatography (HPLC). The highest antibacterial activity was obtained from medium containing 1.5% soluble starch and 2.0% yeast extract, cultured at $30^{circ}C$ for 3.5 days.