This study evaluated the cytotoxicity, antibacterial and physicochemical properties of a new epoxy resin-based endodontic sealer containing calcium-hydroxide Sealer Plus. AH Plus was used as a reference for comparison. Cytotoxicity evaluation was performed according to ISO-10993-5 specifications using MTT assay to check the 3T3 cells viability at 1- to 4-week periods. Antibacterial activity was evaluated using a direct contact test against Enterococcus faecalis. Radiopacity, solubility and flow evaluations were performed according to ISO-6876/2012 specifications. Setting time was assessed following the ANSI/ADA-standard-n.57. The pH level was measured at 3,24,48,72, and 168 hours. Data were statistically analyzed using t-test. The signi?cance level adopted was P<0.05. AH Plus and Sealer Plus showed cytotoxic effects after 24 hours and 1 week of manipulation and become noncytotoxic after 2 weeks. No differences were observed in the cytotoxicity of both tested sealers (P>0.05). Direct contact results revealed that both freshly prepared sealers has antibacterial effects against Enterococcus faecalis. However, after 7 days both sealers had lost much of their antibacterial effects. Although AH Plus presented higher radiopacity and flow than Sealer Plus (P<0.05), both sealers showed minimum required values. No differences were observed in the solubility of both sealers (P>0.05). AH Plus showed a higher setting time when compared to Sealer Plus (P<0.05). AH Plus and Sealer Plus showed a neutral pH during all tested periods (P<0.05). It can be concluded that Sealer Plus showed suitable properties to be used as an endodontic sealer, comparable with those obtained by AH Plus.