Ethanol extract of Myagropsis myagroides (MMEE) has substantial antimicrobial activity against Gram-positive bacteria and some of the yeasts. The minimum inhibitory concentration (MIC) of MMEE ranged from 0.0625 to 0.125 mg/mL for Bacillus subtilis, Listeria monocytogenes, and Clostridium perfringens. Chloroform fraction obtained through liquid-liquid extraction possessed the strongest antimicrobial activity, and was separated by silica gel column chromatography. Among 22 of subfraction, CH4 showed the strongest activity (4-8 times MIC of the crude extract). The antimicrobial activity of CH4 to Staphylococcus aureus was reduced at high concentrations of starch, but the bactericidal effect of CH4 was maintained at 1 and 5% starch model media. The efficacy of CH4 against S. aureus diminished in the presence of >5% beef extract and >1% soybean oil. In acute toxicity test, no mortalities occurred in mice administered 5 g/kg body weight of MMEE over 2 weeks observation.