Objective: To investigate the antimicrobial activity, calcium release, and pH of a new mineral trioxide aggregate endodontic sealer when compared to endodontic sealers containing calcium hydroxide and/or epoxy resin. Method: Specimens were fabricated from MTA Fillapex, Sealer 26, Sealapex, and AH Plus immediately, 24 or 48 hours prior to the tests. The antimicrobial activity against Enterococcus faecalis and Candida albicans was evaluated by the direct contact and the agar diffusion methods. Calcium release was determined by atomic absorption spectrometry. The pH from solutions containing the sealers was measured using a pHmeter. Results: MTA Fillapex, AH Plus and Sealer 26 resulted in partial inhibition of E. faecalis, while Sealapex resulted in the highest bacterium growth. MTA Fillapex and AH Plus also inhibited to a greater extent the growth of C. albicans. None of the investigated sealers could completely inhibit the growth of Enterococcus faecalis and Candida albicans, but influenced microbial growth to different extent. Significantly higher calcium was released from Sealer 26, followed by Sealapex, MTA Fillapex, and AH Plus in a descending order. All investigated sealers resulted in solution with an alkaline pH. Conclusion: The new mineral trioxide aggregate endodontic sealer presented higher antimicrobial activity when compared to the sealers containing calcium hydroxide and/or epoxy resin. As for pH and calcium release, the sealers containing calcium hydroxide resulted in presented the highest values.