The presented work aimed to develop and evaluate a novel hybrid nanocomposite scaffold for bone regeneration, integrating bovine bone-derived nano-hydroxyapatite (nHA), chitosan, and zinc oxide (ZnO) nanostructures. Natural nHA was synthesized via calcination of bovine bone at 1000 °C, while ZnO nanorods were prepared via a sol-gel method. A composite scaffold (75 wt% nHA, 5 wt% ZnO in chitosan matrix) was fabricated. Comprehensive characterization using XRD, FTIR, TEM, and SEM/EDS confirmed the formation of enhanced crystallinity resulting from high-temperature calcination, stoichiometric nHA (~ 30 nm crystallites) and phase-pure ZnO nanorods (30–40 nm diameter). In vivo efficacy was assessed in a rabbit tibial defect model over six weeks. Radiographic and histopathological analyses demonstrated that defects implanted with the composite exhibited enhanced radiopacity and defect filling compared to empty controls. Histology revealed integration of the composite material, bordered by inflammatory and giant cells, with dense collagen deposition. The composite elicited a favorable biological response, supporting its potential as an osteoconductive and antibacterial bone graft substitute that combines the bioactivity of natural HA, the structural benefits of chitosan, and the osteogenic/antibacterial properties of ZnO.