Dual doped calcium apatite has been widely focused as it enhances the osteoconductive property for the possible applications in orthopedic and dental implants. In this work, we investigate the antimicrobial and bioactive properties of cerium/strontium (Ce3+/Sr2+) co-substituted hydroxyapatite (HA) nanoparticles synthesized by sol-gel assisted precipitation method. The structure, morphology, functional groups, photoluminescence, and thermal stability of the developed systems are examined. The comparative studies performed among the pure HA, Sr2+, and Ce3+-substituted HA nanoparticles illustrate higher antibacterial activity with lowered apatite-forming ability and biocompatibility for the Ce3+-substituted HA. However, the Ce3+/Sr2+ co-substituted HA exhibits better biocompatibility, apatite-forming ability, and good antimicrobial properties. Sr2+ ion inclusion leads to better biological properties and compromise the cytotoxic nature of the Ce3+-HA. In addition, the Ce3+/Sr2+-HA nanoparticles prevent thermal decomposition up to 700 degrees C, pointing also toward the possibility of this co-substituted HA in bone implant applications.