In this in vitro study, we assessed the antibacterial efficacy of four endodontic sealers—resin AH26, EndoRez, calcium hydroxide (Apexit), and pure zinc oxide—against Enterococcus faecalis. The agar diffusion test was employed to evaluate the antibacterial efficacy of the sealers in vitro, with distilled water serving as a control. The sealers were prepared following the manufacturer’s instructions and placed in wells of 50 agar plates, each inoculated with 15 samples of Kocuria rhizophila and Staphylococcus aureus. Inhibition zones were assessed after 72, 120, and 168 hours of anaerobic incubation at 37°C for 196 hours. Kruskal-Wallis and Friedman tests were used for data analysis. Positive control plates exhibited bacterial growth in all specified periods. AH26 demonstrated significantly higher antibacterial effectiveness against both bacterium types compared to the other sealers (P<0.01). Pure zinc oxide exhibited moderate antibacterial activity, while Apexit and EndoRez showed the lowest activity against S. aureus and no activity against K. rhizophila. AH26 had the highest antibacterial effect, and EndoRez had the lowest (P<0.05). In terms of inhibiting bacterial growth, the effectiveness of root canal sealers was ranked as follows: AH26 > Pure Zinc Oxide >Apexit/EndoRez.