Objectives: This research evaluated antimicrobial activity of MTA Flow® and assessed whether its bioactivity is preserved in different concentrations and compared to MTA Angelus®. Methods: Methodologies were agar diffusion test (ADT) and direct contact test (DCT) against Enterococcus faecalis and Staphylococcus aureus. Results: In ADT, no cement revealed bioactivity against E. faecalis, while for S. aureus, the lower concentration of MTA Flow® showed significantly lower antimicrobial activity when compared to other MTA Flow® concentrations and MTA Angelus®, after 24 and 48 hours. In ADT, the other concentrations of MTA Flow® were equivalent to that of MTA Angelus® in both exposure times. MTA Flow® and MTA Angelus® had no significant antimicrobial effect against E. faecalis in DCT. On the other hand, with S. aureus, DCT showed that MTA Flow® antimicrobial action was better than MTA Angelus®, at 24 hours for all concentrations. DCT for S. aureus, showed that antimicrobial activity of MTA Flow® and MTA Angelus® has increased over time, demonstrating that antimicrobial property is influenced by the contact time of cements/microorganisms. Conclusion: Thus, none of cements had antimicrobial effect against E. faecalis nether in ADT nor in DCT. All MTA Flow® concentrations had a better performance than MTA Angelus® against S. aureus, in DCT.