The synergistic effects of allicin nanoemulsion (AcN) and ε-polylysine (ε-PL) against Escherichia coli were investigated in this study. The combination of AcN and ε-PL synergistically inhibited the planktonic growth of E. coli, with a low fractional inhibitory concentration index of 0.252. AcN/ε-PL treatment remarkably promoted the agent-cell contacts compared to AcN or ε-PL treatment, as evidenced by the larger cellular size and lower absolute zeta potential value. Analysis of membrane potential, intracellular ATP and superoxide dismutase activity revealed that the co-treatment induced membrane depolarization and intracellular metabolic disorders. Laser scanning confocal microscope, flow cytometer, and scanning electron microscope revealed that the membrane integrity and cell structure were severely degraded. Further, biofilm formation, cluster motility, and mature biofilm of E. coli were disrupted substantially by AcN/ε-PL. Finally, the application of AcN/ε-PL in raw beef preservation verified the synergy. Therefore, AcN/ε-PL can be used as a potential bacteriostatic agent in food preservation.