To develop natural food preservatives, antimicrobial effect of the ethanol extract of leaf mustard against E. coli and S. aureus were examined in terms of compositions and leakage of cellular materials in the microorganisms treated with the extract. No effect of the concentration of ethanol extract on the fatty acid composition of E. coli and S. aureus at logarithmic phase was showen, but the content of palmitic and palmitoleic acid of E. coli slightly increased and decreased, respectively, and the content of palmitic and margaric acid of S. aureus slightly increased, when compared to each control. Ethanol extract did not affect most of the amino acids E. coli and S. aureus at logarithmic phase ; however, some of them(proline, glycine, valine and histidine of E. coli and proline, methionine and histidine of s. aureus) were elevated and some other amino acid(aspartic acid, glutamic acid, tyrosine and arginine of E. coli and aspartic acid, glutamic acid, glycine, alanine and lysine of Staph. aureus) found to be decreased. The amount of cell body protein leaked from E. coli and S. aureus increased to 1.02 and 0.22mg/g cell weight, respectively, as compared to controls. Similarly, the substances with absorbance at 260 nm from E. coli and s. aureus increased to 0.12 and 0.06mg/g cell weight, respectively.