Oral microflora containing microorganisms is responsible for the majority of orodental diseases as well as post-endodontic treatment failure. Even in the ideal root canal treatment cases, a small number of viable bacteria will remain in the dentinal tubule hence come the role of root canal sealers, which must offer and possess some degree of antimicrobial activity alongside adequate flowability to ensure excellent sealing of all lateral dentinal tubule canals to prevent the possibility of future reinfection. The present study aimed to examine the antibacterial efficacy against Enterococcus faecalis (E. faecalis) using an agar diffusion test (ADT) at different time intervals, as well as the flow characteristics of three different root canal sealers: Endofill (Produits Dentaires SA,Vevey, Switzerland), AH Plus (Dentsply De Trey GmbH, Konstanz, Germany), and the newly introduced GuttaFlow2 (Coltène/Whaledent, Altstätten, Switzerland). The antibacterial activity and flow characteristics of three root canal sealers, Endofill, AH Plus, and GuttaFlow2, were tested using ADT at three different time intervals (24 hours, 48 hours, and seven days). For this purpose,E. faecalis strains were used as the pathogen in this study. Flow characteristics were done using the standard flow test protocol recommended for endodontic sealers. Data were analyzed using one-way ANOVA and the Tukey post hoc test, with a p-value < 0.05 considered statistically significant. The results for antibacterial activity showed a statistically significant difference between Endofill and the other groups (p < 0.05). The antibacterial activity of Endofill increased over time from 24 hours to 48 hours and seven days. AH Plus demonstrated antibacterial activity only within the first 24 hours of mixing, while GuttaFlow2 showed no inhibition zones against E. faecalis. Regarding the flow test results, the Endofill group recorded the lowest flow values compared to GuttaFlow2 and AH Plus, which was statistically significant (p < 0.05). There was no statistically significant difference between GuttaFlow2 and AH Plus for flow values. Endofill demonstrated the highest antibacterial activity at all time intervals, while GuttaFlow2 showed no antimicrobial activity. AH Plus exhibited antimicrobial effects only within the first 24 hours of mixing. In terms of flow values, Endofill had the lowest flow, whereas GuttaFlow2 and AH Plus had the highest flow values.