The bacteriostatic action and mechanism of both against Bacillus cereus were studied by using ε-polylysine hydrochloride and nisin alone and in combination. The alkaline phosphatase content, UV absorbing content, pyruvic acid content and SDS-PAGE gel electrophoresis were used as indicators to comprehensively evaluate the antibacterial activity of ε-polylysine hydrochloride and Nisin against Bacillus cereus. The results showed that ε-polylysine hydrochloride and Nisin had a synergistic inhibitory effect on Bacillus cereus. Nisin mainly destroyed cell wall membrane structure and increased the permeability of cell wall membrane, while ε-polylysine hydrochloride mainly affected the growth and metabolism of bacteria by affecting ATPase activity, protein synthesis and expression, and energy metabolism. The combination use of the ε-polylysine hydrochloride and nisin can achieve better bacteriostatic effect at lower concentrations.