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The ethanol extract and n-hexane fraction from Hypericum ascyron L. showed strong growth inhibition at 25 ppm on 5 strains of Listeria monocytogenes for 72 hr at 32oC. The purified substance, H2-5-2 fraction, was isolated by silica gel column and preparative thin layer chromatography from n-hexane fraction of Hypericum ascyron L. The H2-5-2 fraction showed a strong bacteriostatic activity on 5 strains of L. monocytogenes at 10 ppm in tryptic soy broth, and the viable cell was reduced 1 log cycle compared to initial cell number. The n-hexane fraction of Hypericum ascyron L. showed strong growth inhibition at 25 ppm on Bacillus cereus and Staphylococcus aureus, and at 50 ppm on Vibrio parahaemolyticus for 72 hr. The purified antimicrobial substance, the H2-5-2 fraction, was assumed as high unsaturated sterol by 1H-NMR and 13C-NMR. On application test using minced Alaska pollack and ground beef, the n-hexane fraction of Hypericum ascyron L. at the level of 250 ppm was applied at 32oC and 5oC. At 32oC storage condition, the antimicrobial substances did not reduced L. monocytogenes ATCC 19113, meanwhile at 5oC storage condition, L. monocytogenes ATCC 19113 was reduced in viable number.