Aim: The study aimed to evaluate the efficacy of root canal sealer's antimicrobial activity against Enterococcus faecalis. Materials and methods: Root canal sealers with the brands Endoflas FS, AH Plus, and Tubli-Seal EWT were selected. Enterococcus faecalis organisms’ zone of inhibition was measured. Tukey post hoc tests and one-way analysis of variance (ANOVA) were used to compare the data among study groups and within its group. The data were said to be statistically significant with an established p-value less than 0.05. Results: The maximum zone of inhibition (23.20 ± 1.202 mm) was shown by Endoflas FS sealer; AH Plus and Tubli-Seal showed the inhibition zone of (18.42 ± 1.023) and (16.88 ± 0.962) respectively, following Endoflas FS. The ANOVA test showed p < 0.0001, which is highly statistically significant. AH Plus and Endoflas FS showed a statistically significant difference of 0.04 and 0.001 in between groups, and there was no statistically significant difference in the Tubli-Seal group from Tukey post hoc test. Conclusion: The study showed that Endoflas FS sealer has a significant antimicrobial effect against E. faecalis. Clinical significance: In clinical situation, despite perfect chemomechanical root canal preparation, persistence of microorganisms may reinfect the root canal. Therefore, endodontic root canal sealers play a major role in the eradication of bacteria. The polymicrobial nature of endodontic infection plays a main role during the usage of endodontic sealer with antimicrobial agents, which in turn reduces the failure of endodontic treatment.