Aims: The objectives of this research were to show the anti-Escherichia coli O157:H7 effect of fresh (FRMJ) and processed red muscadine (Vitis rotundifolia) juice (PRMJ) and to discern the active compounds responsible for anti-E. coli O157:H7. Methods and Results: Polar and phenolic compounds of FRMJ and PRMJ were analysed by high-performance liquid chromatography. Antibacterial activity of FRMJ, PRMJ, their polar and polyphenol fractions, individual synthetic acids and their mixture with or without sugars were investigated on E. coli O157:H7. FRMJ and PRMJ inactivated (P <= 0.05) 5-log cocktail cells of E. coli O157:H7 within 4 h at 37 degrees . Polar fractions that contained malic, tartaric and tannic acids showed strong antimicrobial activity (P <= 0.05) against E. coli O157:H7. Tannic acid among the synthetic acids showed the highest antimicrobial activity against E. coli O157:H7. Conclusions: FRMJ, PRMJ and their polar compounds showed strong anti-E. coli O157:H7 activity. Significance and Impact of the Study: Earlier findings have failed to show any anti-E. coli O157:H7 effect of grape juice without adding preservatives. Our findings show that red muscadine juice has natural antibacterial substances and suggest that these can be used as active antimicrobial ingredients against E. coli O157:H7 in nonalcoholic beverages.