Introduction: The present study aimed to evaluate the antibacterial effect of the combined use of sodium hypochlorite (NaOCl) and root canal sealers on Enterococcus faecalis biofilms using a dentin infection model. Methods: Cells of E. faecalis were introduced into the dentinal tubules by centrifugation and incubated in brain-heart infusion for 3 weeks. The biofilms in dentin were first subjected to 5% NaOCl or sterile water for 10 minutes followed by an equal thickness of AH Plus (Dentsply International Inc, York, PA), Endosequence BC Sealer (Brasseler USA, Savannah, GA), or MTA Fillapex (Angelus Industria de Produtos Odontologicos S/A, Londrina, Brazil) placed on the root canal wall of the dentin specimens for 7, 30, and 60 days. Gutta-percha and water were used in a similar manner as controls. The proportions of dead and live bacteria inside the dentinal tubules were assessed by confocal laser scanning microscopy and viability staining. Results: The combined use of NaOCl and sealers (30 and 60 days) killed significantly more bacteria than NaOCl or sealers alone (P < .05). NaOCl + MTA Fillapex was the most effective antibacterial combination by killing 83% bacteria in dentin tubules in 60 days. Thirty and 60 days of exposure to the sealers resulted in significantly more dead bacteria in dentin biofilms than 7-day exposures (P < .05). Conclusions: The placement of root canal sealer after NaOCl treatment enhanced antibacterial effects against E. faecalis in the dentinal tubules. Little additional effect was obtained after 30 days of exposure to sealers.