The solvent extracts of Euphorbia helioscopia, which were extracted by using several solvents with different polarities, were prepared for utility as natural preservatives. The E. helioscopia extract by 80% ethanol was sequentially fractionated with n-hexane, dichloromethane, ethylacetate, and butanol. In order to effectively screen for a natural preservatives agent, we first investigated the antioxidant activities such as DPPH radical scavenging capacity, superoxide radical scavenging capacity, and xanthine oxidase inhibitory activity of the E. helioscopia extracts. By the screening system, we found that ethylacetate fraction had the strongest antioxidant activity in a dose-dependent manner. The antimicrobial activities and cell growth inhibition were investigated for each strain with the different concentrations of E. helioscopia extracts. Antimicrobial activities were shown in ethylacetate fraction of E. helioscopia; however, ethanol, butanol and water fractions showed weak antimicrobial activity against the tested microorganisms. Among the five fractions, ethylacetate fraction showed the highest antimicrobial activities against microorganisms tested, such as Bacillus sublitis, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella Enteritidis and Salmonella Typhimurium. The polyphenol content from ethanol, n-hexane, dichloromethane, ethylacetate, butanol, and water fractions were 207.46 mg/g, 45.45 mg/g, 138.23 mg/g, 678.02 mg/g, 278.91 mg/g, and 63.76 mg/g, respectively. There seems to be a close relationship between antioxidant activities, and antimicrobial activities and polyphenol content in natural plant. From these results, it is suggested that E. helioscopia could be used for the ethylacetate fraction and could be suitable for the development of a food preservative.