The objective of this study was to prepare hydroxyapatite (HA) with potential antibacterial activity against gram-negative and gram-positive bacteria by incorporating different atomic ratios of Cu2+ (0.1–1.0%), Mg2+ (1.0–7.0%), and Zn2+ (1.0–7.0%) to theoretically replace Ca2+ ions during the hydrothermal synthesis of grown precipitated HA nanorods. This study highlights the role of comparing different metal ions on synthetic nanoapatite in regulating the antibacterial properties and toxicity. The comparisons between infrared spectra and between diffractograms have confirmed that metal ions do not affect the formation of HA phases. The results show that after doped Cu2+, Mg2+, and Zn2+ ions replace Ca2+, the ionic radius is almost the same, but significantly smaller than that of the original Ca2+ ions, and the substitution effect causes the lattice distance to change, resulting in crystal structure distortion and reducing crystallinity. The reduction in the length of the nanopatites after the incorporation of Cu2+, Mg2+, and Zn2+ ions confirmed that the metal ions were mainly substituted during the growth of the rod-shape nanoapatite Ca2+ distributed along the longitudinal site. The antibacterial results show that nanoapatite containing Cu2+ (0.1%), Mg2+ (3%), and Zn2+ (5–7%) has obvious and higher antibacterial activity against gram-positive bacteria Staphylococcus aureus within 2 days. The antibacterial effect against the gram-negative bacillus Escherichia coli is not as pronounced as against Staphylococcus aureus. The antibacterial effect of Cu2+ substituted Ca2+ with an atomic ratio of 0.1~1.0% is even better than that of Mg2+- and Zn2+- doped with 1~7% groups. In terms of cytotoxicity, nanoapatites with Cu2+ (~0.2%) exhibit cytotoxicity, whereas Mg2+- (1–5%) and Zn2+- (~1%) doped nanoapatites are biocompatible at low concentrations but become cytotoxic as ionic concentration increases. The results show that the hydrothermally synthesized nanoapatite combined with Cu2+ (0.2%), Mg2+ (3%), and Zn2+ (3%) exhibits low toxicity and high antibacterial activity, which provides a good prospect for bypassing antibiotics for future biomedical applications.