Endodontic sealers with antibacterial capability play an important role in preventing reinfection of an endodontically treated root canal and improving the long-term success of root canal treatment. However, current endodontic sealers rapidly lose their antibacterial properties after fixation. In this work, we designed and synthesized quaternized mono-dispersed bioactive nanospheres as a potential substrate for the development of a long-term antibacterial endodontic sealer with excellent cytocompatibility and biocompatibility. First, mono-dispersed silica-based bioactive glass nanospheres (SBG-NS) were prepared via a modified sol-gel process. Next, a series of quaternary ammonium methacrylate salts (QAMs) with broad antibacterial spectra were synthesized and grafted onto the surfaces of the SBG-NS via a two-step coupling approach. The antibacterial effect of the quaternary ammonium polymethacrylate (QAPM)-containing SBG-NS (SBG-QAPM) against persistent microorganisms associated with infected root canals was evaluated using a direct contact test. Evaluations of the SBG-QAPM cytocompatibility and biocompatibility were performed using LIVE/DEAD staining, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2, 5-tetrazoliumbromide (MTT) assay, and a calvarial implantation model. The results showed that the SBG-QAPMs had the strongest long-term antibacterial effect against the Enterococcus faecalis, Streptococcus mutans, and Streptococcus sanguis during the study period, the best cytocompatibility, and the lowest systemic inflammation compared to three commercial products: ProRoot MTA, Endomethasone C, and AH Plus. In addition, the SBG-QAPMs showed excellent stability in aqueous solution. This work indicates that the SBG-QAPMs are promising substrates for the development of long-term antibacterial endodontic sealers.