This study evaluated the antibacterial effect of the combination of ɛ-polylysine (ɛ-PL) and nisin against Enterococcus faecalis strains. The combination of ɛ-PL and nisin showed synergistic antibacterial activity against three Enterococcus strains. Scanning electron microscopy and a membrane permeability assay revealed that the combined treatment with ɛ-PL and nisin synergistically damaged the cell morphology of E. faecalis strain R612Z1 cells. Both ɛ-PL and nisin can dissipate the transmembrane electric potential of E. faecalis R612Z1 cells, but these peptides did not affect the transmembrane pH gradient. The combination of ɛ-PL and nisin can produce a high reactive oxygen species level in E. faecalis R612Z1 cells. The results indicated that the uptake of ɛ-PL into cells was promoted through nisin and that the combination of ɛ-PL and nisin could produce a high reactive oxygen species level in E. faecalis R612Z1 cells, leading to cell growth inhibition.