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This study was conducted to examine the antimicrobial activity of Perilla frutescens var. acuta leaf fractions extracted with a mixture of ethanol and water. The Ca and Mg contents of the leaf were 595.75 mg% and 467.0 mg%, respectively, and they were the highest among all of the tested minerals. The extract yield increased with the content of water in the extraction solvent. Antimicrobial activity against Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa was found in the 50, 70 and 95% ethanol extracts. Of the various fractions extracted from the mixture of ethanol and water, the ethyl acetate fraction showed antibacterial activity against all microorganisms tested in this experiment, and the antibacterial activity of ethyl acetate fraction from the water extract was the strongest. The phenol and flavonoid content in the ethyl acetate fraction showed no correlation with the concentration of ethanol in the extract solvent; however, their contents were higher in the 30% ethanol and water extraction which the antimicrobial activity of the extract was the strongest.