Bioactive materials are playing an important role in sealing apical root canals of teeth, inducing bone/cementum tissue regeneration and inhibiting bacterial viability. Conventional Ca(OH)2 materials have limitations for filling apical root canals of teeth due to their low mineralization ability and potential cytotoxicity. The aim of this study is to prepare bioactive mesoporous calcium–silicate (MCS) nanoparticles for the potential application of filling an apical root canal of a tooth. The mesoporous structure, specific surface area, pore volume and morphology of MCS particles were characterized. The apatite-mineralization ability, in vitro osteogenesis, drug delivery and antibacterial properties were further investigated. The results showed that MCS nanoparticles (around 100 nm) with high specific surface area and pore volume were successfully prepared by a facile template method. The prepared MCS could be easily injected to fill the apical root canal of a tooth. MCS nanoparticles induced apatite-mineralization in DMEM solution, did not show cytotoxicity, and their ionic products could stimulate the proliferation of periodontal ligament cells (PDLCs). In contrast, conventional Ca(OH)2 materials did not induce mineralization and showed significant cytotoxic effects on PDLCs. Furthermore, MCS extracts at low concentrations (12.5 and 25 mg mL−1) induced higher ALP activity of PDLCs than those at high concentrations (50 and 100 mg mL−1). In addition, MCS extracts significantly stimulated osteogenic gene expression (OPN, ALP and OCN) of PDLCs compared to a blank control, indicating the excellent osteostimulation property of MCS. MCS nanoparticles could be used for loading the antibiotic ampicillin due to their mesoporous microstructures, and the loaded ampicillin in MCS nanoparticles could be released with a slow and sustained release profile. Moreover, it was found that pure MCS nanoparticles revealed antibacterial effects, while the delivery of ampicillin from MCS nanoparticles further inhibited bacterial viability. Therefore, the results suggest that MCS nanoparticles are an advanced biomaterial with multiple functions for filling the apical root canal of a tooth due to their unique nanostructure, injectability, apatite-mineralization, osteostimulation, drug-delivery and antibacterial properties.