Background: This study aimed to compare the antibacterial effects of calcium hydroxide, curcumin, and Aloe vera as an intracanal medicament on 6-week-old Enterococcus faecalis biofilm. Materials and Methods: In this in vitro study, the solution containing E. faecalis ATCC® 29212™ was inserted into the canals of 72 single-rooted teeth to produce biofilm. The samples were divided into four groups, and the antibacterial agent as an intracanal drug was used for 1 week. Calcium hydroxide, curcumin, and A. vera were used as intracanal medicaments in three groups, respectively, and the fourth group was irrigated with normal saline. The collected debris was cultured by spread plate method for the bacterial count by colony count machine, and the number of bacteria in each sample per ml was reported in colony-forming unit per ml (CFU/ml). The data were analyzed using SPSS software. KruskalWallis and MannWhitney U-tests were used for comparison of CFU/ml between the study groups. P <0.05 was considered significant. Results: The mean CFU/ml in the groups of calcium hydroxide, curcumin, and A. vera were 749.44, 630.55, and 1529.16, respectively. Compared with the control group, curcumin, calcium hydroxide, and A. vera showed 99.5%, 99.41%, and 98.79% antimicrobial effects, respectively. All three groups were significantly effective than the control group (P = 0.023, P = 0.023, and P = 0.024, respectively) but were not significantly different from each other (P = 0.057). Conclusion: All three groups showed significant antibacterial activity compared to the control group, curcumin had the most significant effect, followed by calcium hydroxide and A. vera. Therefore, herbal materials can be considered safe alternatives to synthetic medicaments for intracanal usage. [ABSTRACT FROM AUTHOR]