ε-Polylysine (ε-PL) is a cationic antimicrobial peptide, which easily forms complexes with food polyanions to weaken its antibacterial activity. A whey protein-ε-PL complex delivery system was found to be able to solve this problem. This study investigated the antimicrobial activity of the complexes and their mechanism against Gram-positive bacteria. The minimal inhibitory concentration of the complexes with different ε-PL contents against Staphylococcus aureus and Bacillus subtilis were 19.53-31.26 and 3.90-7.81 μg/mL, respectively, which were similar to free ε-PL. Furthermore, the whey protein-ε-PL complexes had a strong bactericidal effect on Bacillus subtilis. The inhibition zone diameters of the complexes against Staphylococcus aureus and Bacillus subtilis containing 5000 μg/mL of ε-PL were 14.14 and 16.69 mm, respectively. The results of scanning electron microscopy showed that the complexes could destroy the cell membrane structure in Bacillussubtilis, resulting in holes on the surface, but not in Staphylococcus aureus. The results of molecular dynamics simulation showed that under electrostatic interaction, the complexes captured the phospholipid molecules of the bacterial membrane through the hydrogen bonds. Parts of the ε-PL molecules of the complexes were embedded in the bilayer membrane, and parts of the ε-PL molecules could penetrate the bilayer membrane and enter the bacterial internal environment, forming holes on the surface of the bacteria. The antibacterial results in fresh meat showed that the whey protein-ε-PL complexes could reduce the total mesophilic and Staphylococcus aureus counts. This study on the antibacterial activity mechanism of whey protein-ε-PL complexes could provide a reference for the application of ε-PL in protein food matrices.