Statement of the Problem: Hydrogen peroxide (H2O2) has been suggested to be used in sequence or in combination with chlorhexidine (CHX) to enhance the antibacterial activity against Enterococcus faecalis, but there is no research in the literature on the safety and effectiveness of this irrigation protocol.Purpose: This study aimed to assess the cytocompatibility and antibacterial activity of different concentrations of CHX combined with H2O2 in comparison with the activity of 5.25 and 2.5% sodium hypochlorite (NaOCl).Materials and Method: Different concentrations of H2O2 (10, 5, 3 and 1%) were exposed to the PDL cells. Then, the solution with minimal cytotoxicity was selected (3% H2O2). The cytocompatibility and antibacterial activity of 0.1, 0.2, 1 and 2% CHX combined with 3% H2O2 were evaluated and compared with 5.25 and 2.5% NaOCl. The differences in the mean viability of PDL cells were evaluated by one-way ANOVA. Kruskal-Wallis and post-hoc Dunn's tests were adopted to compare the antibacterial activity of the solutions against E.faecalis.Results: The viability of PDL cells was lower when treated with 5.25 or 2.5% NaOCl than all combinations of CHX and H2O2.There was no significant difference in the antibacterial activity of the solutions against E.faecalis, except for the 0.1% CHX + 3% H2O2 combination, which had significantly lower efficacy than other groups.Conclusion: All combinations of CHX and H2O2 (used in this study)except 0.1% CHX + 3% H2O2 were efficient irrigants against planktonic E.faecalis and had a better cytocompatibility with PDL cells than 5.25 and 2.5% NaOCl.