The aim of the present study is to examine the function of the two significant biological cations, Zn2+ and Sr2+, when co-doped in the structure of hydroxyapatite (HA). Six samples with different Zn2+ and Sr2+ concentrations were prepared by a hydrothermal process that includes the addition of Zn2+ and Sr2+ containing precursors to replace Ca2+ partially in the HA structure. The obtained nanomaterials were examined via XRD, ICP, FTIR, Raman spectroscopy, FESEM, TEM, and TGA for the investigation and confirmation of co-doping, crystallinity, phase purity, lattice parameters, elemental compositions, particle morphology, and thermal stability respectively. Interestingly the Zn2+ substitution increase with Sr2+ co-doping and the apatite structure going disappear up to 20%Zn10%Sr or 10%Zn20%Sr in the solution. Furthermore, in vitro cell viability even at high Zn2+ concentration (up to 6 wt %), MC3T3-E1 cells proliferated at a high rate on co-substituted HA materials, confirming that Sr2+ offset the cytotoxic effect of Zn2+ and increased the biocompatibility of the material. Moreover, the antibacterial effects of co-substituted HA against pathogen bacterial strains (E.coil and S.aureus) were also significantly increased. Overall results demonstrated that HA co-substituted by Zn2+ and Sr2+ at (10%Zn10%Sr-HA) is a potential material for hard tissue scaffolds.