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Nisin, a broad-spectrum bacteriocin, has recently been highlighted for its biomedical applications. To date, no studies have examined the antimicrobial and antibiofilm properties of high-purity (>95%) nisin (nisin ZP) on Enterococcus faecalis and biofilms formed by this species. We hypothesize that nisin can inhibit E. faecalis and reduce biofilm biomass, and combinations of nisin and sodium hypochlorite (NaOCl) will enhance the antibiofilm properties against E. faecalis biofilms.Using broth cultures, disc diffusion assays, and biofilm assays, we examined the effects of nisin on various E. faecalis growth parameters and biofilm properties (biovolume, thickness, and roughness). Confocal microscopy was used in conjunction with Imaris and Comstat2 software (Kongens Lyngby, Copenhagen, Denmark) to measure and analyze the biofilm properties.Nisin significantly decreased the growth of planktonic E. faecalis dose dependently. The minimum inhibitory concentrations against E. faecalis strains OG-1 and ATCC 29212 were 15 and 50 μg/mL, and the minimum bactericidal concentrations were 150 and 200 μg/mL, respectively. A reduction in biofilm biovolume and thickness was observed for biofilms treated with nisin at ≥10 μg/mL for 10 minutes. In addition, the combination of nisin with low doses of NaOCl enhanced the antibiofilm properties of both antimicrobial agents.Nisin alone or in combination with low concentrations of NaOCl reduces the planktonic growth of E. faecalis and disrupts E. faecalis biofilm structure. Our results suggest that nisin has potential as an adjunctive endodontic therapeutic agent and as an alternative to conventional NaOCl irrigation.Nisin ZP decreases the growth of planktonic Enterococcus faecalis dose dependently.Nisin ZP reduces E. faecalis biofilm structures.Combinations of nisin with low doses of NaOCl enhances the antibiofilm properties of both agents.Nisin has potential as an adjunctive or alternative therapeutic agent to the NaOCl solution.