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The aim of this study was to evaluate the efficacy of chitosan nanoparticles (CNPs) and ethanolic propolis extract (EPE) incorporated into a calcium hydroxide paste (Ca[OH]2) to kill bacterial biofilms.Human root canal dentin was infected with Enterococcus faecalis for 21 days and also intraorally for 48 hours followed by incubation in brain-heart infusion for 48 hours to standardize biofilm growth. Ca(OH)2 pastes associated or not with CNPs or EPE were tested on biofilms for 7 and 14 days. Distilled water was used for control purposes. After the treatment procedures, microbiological analysis was performed to determine the reduction in E. faecalis colonies. Confocal microscopy was used to determine the percentage of cell viability in polymicrobial biofilms before and after the exposure to the experimental intracanal medications.All experimental pastes were able to significantly reduce the E. faecalis colony-forming units (CFU) after 7 or 14 days (P < .05). However, the CFU reduction was significantly improved when CNPs were incorporated into the Ca(OH)2 paste (P < .05). The multispecies biofilms treated with Ca(OH)2 showed similar percentages of bacterial viability to the control regardless of the exposure time (P > .05). The viable cell count significantly dropped in the Ca(OH)2/CNPs groups for both 7 and 14 days (P < .05), whereas the Ca(OH)2/EPE groups were only effective in eliminating bacteria during the first 7 days of treatment (P < .05).Incorporating CNPs into pastes of Ca(OH)2 could potentially be beneficial when using interappointment intracanal medications because of their ability to kill bacteria in short- and long-term exposure.We evaluated the antibacterial ability of CNPs and EPE incorporated into Ca(OH)2.Ca(OH)2 /CNPs was the most effective association to kill bacteria.Ca(OH)2 /EPE lost its antibacterial properties on polymicrobial biofilms over time.Ca(OH)2 was not able to significantly reduce the bacterial charge in oral biofilms.