In order to develop a natural food preservative, the root bark of Morus alba was extracted with several solvents, and then antimicrobial activity was investigated. The optimum extracting condition for the antimicrobial substance from the sample, minimum inhibitory concentration (MIC) of the extracted substance against microorganisms were also examined. The antimicrobial activity of the ethanol extract form the sample was stronger than those of the extracts by the other solvents such as water, methanol. ethyl acetate and acetone. The optimum extracting condition for antimicrobial substance from the sample was shaking extraction twice for 5 hours at room temperature in case of 7 times of absolute ethanol added to the crushed root bark of Morus alba. The ethanol extract from the root bark of Morus alba had strong B. cereus, L. monocytogenes and S. aureus. Especially, Bacillus species was the most susceptible to the extracted substance. The ethanol extract showed antimicrobial activity against Gram negative bacteria(MIC, 160~1600 ug/ml) and yeasts(MIC, 1600 ug/ml) such as C. albicans and S. acidifaeciens. The extract also showed growth inhibition against molds such as A. niger, A. parasiticus, A versicolar and T. viride.