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Objectives: Antibacterial activity against endodontic pathogens is a desirable feature for root canal sealers. The objective of this study was to compare the antibacterial effect of three resin-based endodontic sealers (AH26, Adseal, and Beta RCS) against Enterococcus faecalis in vitro. Materials and Methods: The antibacterial properties of the sealers were assessed against E. faecalis using agar diffusion test (ADT) for fresh state (N=10) and direct contact test (DCT) for freshly-mixed and set states of the materials (N=10). In ADT, the diameter of the zones of inhibition was measured after 24h of contact. In DCT, the colony-forming units of the bacteria were counted after 30 minutes and 180 minutes of exposure. The results were analyzed with two-way ANOVA and independent sample t-test. P<0.05 was considered significant. Results: Regarding DCT results, all test materials indicated an antibacterial effect, both in freshly-mixed and set states. The highest antibacterial effect was related to Adseal, whereas the lowest was observed in Beta RCS. There was a significant difference between all study groups (different sealers, setting states, and contact times; P<0.001), except for freshly-mixed AH26 and Adseal at 180 minutes (P>0.05). According to ADT, AH26 and Adseal represented the widest and the smallest inhibition zones, respectively (P<0.001). Conclusion: Within the limitations of this in vitro study, AH26, Adseal, and Beta RCS showed antibacterial effects against E. faecalis in both freshly-mixed and set states. The antibacterial effect increased over time in all of the studied sealers.