The aim of this study is to evaluate the antimicrobial activity of the fast-setting iRoot Fast Set Root Repair Material (iRoot FS), Mineral trioxide aggregate (MTA) and Biodentine. The materials were freshly mixed or set for 1 and 7 days to conduct the agar diffusion test, direct contact test and carry-over effect test against E. faecalis and P. gingivalis, and the pH values were also measured. The data were analyzed by an analysis of variance and one-way ANOVA or Dunnett’s T3 test, and the Tukey’s post hoc test for multiple comparisons (α = 0.05). In the direct contact test, all three materials showed good antibacterial activity after setting for 20 min. The antibacterial properties of the three materials decreased with the increase of setting time ( p < 0.05). The suspension of all the three materials showed high pH values (11–12) and no significant difference was observed ( p > 0.05). With the extension of setting time, the pH of iRoot FS and Biodentine slightly decreased ( p < 0.05). Fresh iRoot FS, Biodentine, and MTA killed E. faecalis and P. gingivalis effectively, but their antimicrobial effect decreased after 24 h, and distinctly decreased after 7 days after mixing. iRoot FS, Biodentine, and MTA showed a tendency of alkalinity during this 7-day experiment.