To solve the high instances of failure caused by endodontic reinfection, herein, an improved root filling material was produced to meet the multi-functional demand of sealers for root canal therapy. In this study, polyurethane (PU)-based nanocomposites were prepared by loading bismuth oxide, hydroxyapatite and antibacterial agents, namely Ag3PO4 and ZnO nanoparticles, which were named CP-Ag and CP-Zn sealers, respectively. A parallel biological evaluation at bacterial and cellular levels was performed to determine the fate of the different components of the PU-based sealers. Furthermore, the composition of sealers was quantified by screening their antibacterial activity and apoptotic factors, considering the potential toxicity of the nanoparticles and high dosage of metals. The in vitro optimization investigation was conducted systematically against Streptococcus mutans and Staphylococcus aureus, including bacteriostatic and dynamic tests, and the expression of the B-cell lymphoma-2 gene family and caspase proteases in the mitochondria-mediated apoptotic pathway was evaluated using the commercial AH Plus® and Apexit® Plus sealers for comparison. Additionally, the physical properties and sealing ability of sealers were assessed. The results showed that all PU-based sealers could meet the requirements of ISO 6876:2012 for root canal sealing materials. Based on the evaluation system, CP-Zn sealers expressed longer lasting antibacterial activity and lower toxic effect on cells compared to CP-Ag sealers. Especially, the CP-Zn5 sealer exhibited selective antimicrobial efficacy and hypo-toxicity, which were better than that of the two commercial sealers. According to the two-dimensional and three-dimensional methods, the good sealing ability of the CP-Zn5 sealer is the same as the excellent filling characters of AH Plus, which adapts to irregular root canals.