The solvent extracts of Oenothera laciniata, which were extracted by using several solvents with different polarities, were prepared for utility as a natural preservative. The O. laciniata extract by 80% ethanol was sequentially fractionated with n-hexane, dichloromethane, ethylacetate, and butanol. The antibacterial activities and cell growth inhibition were investigated on each strain with the different concentrations of O. laciniata extracts. Antibacterial activities were shown in ethanol, ethylacetate, and butanol fraction of O. laciniata. However n-hexane, dichloromethane and water fraction showed weak antibacterial activity against the tested microorganisms. Among the five fractions, ethylacetate fraction showed the highest antibacterial activities against microorganisms tested, such as Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Enteritidis and Salmonella Typhimurium. The polyphenolic compounds widely occurring in the traditional medicine plants have been reported to possess high antibacterial activity. The polyphenolic compounds from ethanol, n-hexane, dichloromethane, ethylacetate, butanol, and water fraction were 63.96 mg/g, 8.49 mg/g, 28.11 mg/g, 172.64 mg/g, 114.56 mg/g, and 34.91 mg/g, respectively. There are some relationships between antibacterial activity and polyphenol content in natural plant. The ethylacetate fraction could be suitable for the development of a food preservative.