Polyphenols are predominantly secondary metabolites in muscadine grapes, playing an important role in the species' strong resistance to pests and diseases. This study examined the above property by evaluating the antioxidant, antibacterial, and antibiofilm activities of muscadine polyphenols against selected foodborne pathogens. Results showed that antioxidant activity for different polyphenols varied greatly, ranging from 5 to 11.1 mmol Trolox/g. Antioxidant and antibacterial activities for polyphenols showed a positive correlation. Muscadine polyphenols exhibited a broad spectrum of antibacterial activity against tested foodborne pathogens, especially Staphylococcus aureus (MIC = 67-152 mg/L). Muscadine polyphenols at 4 × MIC caused nearly a 5 log10 CFU/mL drop in cell viability for S. aureus in 6 h with lysis, whereas at 0.5 × MIC they inhibited its biofilm formation and at 16 × MIC they eradicated biofilms. Muscadine polyphenols showed synergy with antibiotics and maximally caused a 6.2 log10 CFU/mL drop in cell viability at subinhibitory concentration.