The complex structure of the root canal system and microbial resistance increase the difficulty of endodontic treatment and the development of root canal sealers with good antibacterial and physicochemical properties is the key to treat refractory root canal infection. In the present study, a novel premixed root canal sealer containing trimagnesium phosphate (TMP), potassium dihydrogen phosphate (KH2PO4), magnesium oxide (MgO), zirconium oxide (ZrO2), and a bioactive oil phase was developed, and the physicochemical properties, radiopacity, antibacterial activity in vitro, anti-biofilm ability and cytotoxicity were investigated. MgO significantly improved the anti-biofilm ability and ZrO2 enhanced the radiopacity of the premixed sealer, and they had an obvious adverse effect on other properties. In addition, this sealer has advantages such as easy-to-use design, storabality, good sealing ability and biocompatibility. Therefore, this sealer has high potential for use in treating root canal infection.