Punicalagin (PUN), as a natural antimicrobial agent, is the most abundant phenolic compound in pomegranate peel. In this study, the bactericidal effect of PUN concentrations, high pressure conditions and their combination on E. coli O157: H7 were investigated. The bacteria were suspended in physiological saline solution with PUN concentrations of 1–3 mg/mL and treated by high pressures of 100–300 MPa for 2–10 min. The synergistic effect of the combination treatment was demonstrated with the maximum enhancement value of 4.10. Cell morphology changes, membrane permeability enhancement and swimming motility inhibition were observed in the combination treatment. Adenosine, adenine, cyclic GMP in purine metabolism and glutathione in glutathione metabolism pathways were differential metabolites with content changes, which were related with treatment methods (HPP or PUN) but not the degree of bacteria inactivation. This study provided a more effective and innovative method for killing E. coli in simulation system. HPP was the most commercialized emerging non-thermal processing technology and PUN was the natural antibacterial agent from pomegranate peel waste. The combination of HPP and PUN had synergistic bactericidal effect on E. coli with less processing intensity, which was very suitable for the products those contained thermosensitive components with maximum sensory characteristics preserved and less preservative added.