The present study aims to synthesize biocompatible and bioactive lanthanum (La3+)/strontium (Sr2+) dual ion doped hydroxyapatite (HA) nanomaterials by sol-gel method. The discrete substitution of La3+ and Sr2+ in pure HA enhances the osteoconductivity. The co-substitution of various La3+ concentrations (0.03, 0.06 and 0.1 M) regulates the physical and In Vitro properties. The study also investigates the effect of La3+/Sr2+ substituents on the crystalline property, microstructure, photoluminescence and In Vitro bioactivity of HA samples. La3+/Sr2+ co-substitution decreases the crystallite size of HA without any significant distortion of the crystal structure. In addition, the dual ions doping influences nanoparticles morphology by reducing the particle size from 75 to 20 nm. The In Vitro bioactivity tests for the La3+/Sr2+ co-substituted HA confirm the osteoconductive boneapatite generating capacity. Bactericidal tests against Staphylococcus aureus and Pseudomonas aeruginosa stains show better resistivity of La3+/Sr2+-HA samples. To authenticate the biocompatibility and antimicrobial activity of the synthesized La3+/Sr2+ dual doped HA nanoparticles for bone implant applications, different tests like cell viability and toxicity were conducted using human lung A549 cells.