Hydroxyapatite is naturally found in bones and it is used to deliver antibacterial agents such as chlorhexidine and gentamicin to bones. This study covered the development of hydroxyapatite and nickel (Nix = 0.3,0.5) substituted hydroxyapatite nanoparticles by co-precipitation method. The characterization of nickel (Nix = 0.3,0.5) substituted hydroxyapatite was done by X-ray diffraction, Infra-Red studies and Scanning Electron Microscopy. This study also includes in vitro controlled drug delivery of nickel (Nix = 0.3,0.5) substituted hydroxyapatite nanoparticles which were loaded with widely consumed drug, ciprofloxacin. The antibacterial and antioxidant activity of nickel (Nix = 0.3,0.5) substituted hydroxyapatite was also checked. Our results indicated that nickel substitution in hydroxyapatite increased the percentage of drug (ciprofloxacin) loading up to 96% and cumulative drug release up to 95% in a tunable controlled mode of action. Free radical scavenging or antioxidant comparative analysis of pure and cationic substituted hydroxyapatite further revealed significant enhancement in iron-chelating potential. Nonetheless, nickel (Nix = 0.3,0.5) substituted hydroxyapatite nanoparticles loaded with ciprofloxacin showed strong antibacterial activity (22 mm and 24 mm zone of inhibition against E. coli (ATCC 35218) and P. aeruginosa (ATTC 27853) strains respectively). This development allows the researchers in the area to get a prompt comparison and sort out the best solutions for the cationic substitution of hydroxyapatite-based materials and enable the development of multi-functional biomedical drug delivery systems and designs for the most commonly used antibiotic molecules.