The ethanol extract of leaf mustard(Brassica juncea) exhibiting high antimicrobial activities was fractionated in the order of hexane, chloroform, ethylacetate and butanol fractions to test antimicrobial activity. The highest antimicrobial activity for the bacteria tested was found in the ethylacetate fraction, but a lesser extent in the butanol fraction. In contrast to antimicrobial activity for the bacteria, both ethylacetate and butanol fractions showed weak antimicrobial activity for yeasts. Unknown compound A in the ethylacetate fraction which exhibited a strong antimicrobial activity was isolated by silica gel column chromatography and HPLC, and exhibited 9 times more antimicrobial activity than the ethylacetate fraction.