Technical Abstract: Hot and cold water-soluble muscadine (Vitis rotundifolia) seed extracts and their polar and polyphenol fractions from two Muscadine cultivars (‘Ison’, purple and ‘Carlos’, bronze) were investigated for their inhibition of Enterobacter sakazakii. The heat treatment on each seed extract not only increased total phenolics and tannic acid but also enhanced antimicrobial activity against two strains of E. sakazakii. Within 1 h, all seed extracts reduced an initial population ( 6 log CFU/mL) of E. sakazakii to a non-detectable level (minimum detection limit, 10 CFU/mL). Regardless of extraction method and cultivar, only the polar fractions which contained malic, tartaric and tannic acids showed antimicrobial activity against two strains of E. sakazakii. The polyphenol fractions which contained gallic acid, catechin, epicatechin, ellagic acid and pigments showed slight inhibition against E. sakazakii. Results showed that water-soluble muscadine seed extracts (pH 3.3–3.78) contained strong antimicrobial inhibitors against E. sakazakii while acidified peptone water (pH 3.3) did not show any antimicrobial activity.