<p>In recent years, the synthesis of metal-doped hydroxyapatite nanoparticles has been extensively studied and recognized as a nontoxic and efficient method applicable in the biomedical field. In this study, we synthesized and characterized cationic-substituted hydroxyapatite to investigate its dual action in anticancer and antibacterial therapies, which showed efficient cytotoxicity of doped hydroxyapatite (HAp) on the human liver carcinoma cell line (Hep G2) and enhanced antibacterial effect on <i>Staphylococcus aureus</i> and <i>Micrococcus luteus</i> bacterial strains. Nano-hydroxyapatite was prepared by co-precipitation technique with doping of various concentrations (x = 0, 0.7, 0.9) of nickel Ni²⁺ and zinc Zn²⁺ with the chemical formula (Ca)_10−xM_x(PO₄)₆(OH)₂ where M = Ni²⁺, Zn²⁺. Results from the antibacterial investigation specified that Ni²⁺ and Zn²⁺ doped hydroxyapatite have strong 50 ± 0.4&#xa0;mm and 60 ± 0.5&#xa0;mm inhibition zones, especially against multidrug-resistant gram-positive <i>S. aureus</i>. The zeta potential of pure hydroxyapatite (HAp), nickel-doped hydroxyapatite (Ni-HAp), and zinc-doped hydroxyapatite (Zn-HAp) nanocomposites were − 3.23 mV, 2.995 mV, and 6.9 mV, respectively. The charge shift from a negative to a positive zeta potential due to cationic substitution indicated substantial changes in the surface interaction potential of the doped hydroxyapatite nanoparticles at particular conditions, which validated enhanced antibacterial and anticancer activity through experimental investigations. In vitro, cytotoxicity screening of pure HAp, Ni²⁺, and Zn²⁺ doped hydroxyapatite against Hepatocellular carcinoma HepG2 cells differentiated and interpreted. The release of nickel and zinc ions with calcium and phosphate enhanced the antibacterial and anticancer activity of doped hydroxyapatite. Zinc-doped hydroxyapatite has a better 69.98 ± 5.79% potential for cytotoxicity anti-cancerous activity evidenced by cell viability studies.</p>