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.