The antimicrobial effect of each fraction after fractionation of an ethanol extract of leaf mustard was examined in terms of nucleic acid, chloroform, ethylacetate, and butanol. The ethylacetate fraction, which showed the strongest level of antimicrobial effect among the different ethanol extract fractions of leaf mustard, was isolated and purified using silica gel column chromatography and HPLC, respectively, to obtain a single antimicrobial substance called KLM-1. The antimicrobial effect of this substance was 10 times higher than that of the ethylacetate fraction. A further study is on the way to confirm the structure of the antimicrobial substance KLM-1 through LC/Mass and NMR.