Hydroxyapatite (HA) is a bioceramic material widely used as a bone biomimetic substitute and can be synthesized by biomineralization, according to which HA nanoparticles are formed on a polymer template. Nevertheless, little is known about the effect of ion doping and biomineralization on cell metabolism, oxidative stress, and DNA damage. In the present contribution, we report on synthesizing and characterizing biomineralized chitosan as a polymer template with HA nanoparticles doped with magnesium (MgHA) and iron ions (FeHA). The physical-chemical and morphological characterization confirmed the successful synthesis of low crystalline ions-doped HA nanoparticles on the chitosan template, whereas the biochemical activity of the resulting nanoparticles towards human osteoblasts-like cells (MG63 and HOBIT) was investigated considering their effect on cell metabolism, proliferation, colony formation, redox status, and DNA damage extent. Data obtained suggest that particles enhance cell metabolism but partially limit cell proliferation. The redox status of cells was measured suggesting a slight increase in Reactive Oxygen Species production with chitosan biomineralized with iron-doped HA, whereas no effect with magnesium-doped HA and no effect of all formulations on the oxidation level of Peroxiredoxin. On the other hand, DNA damage was investigated by COMET assay, and expression and foci γH2AX. These latter tests indicated that HA-based nanoparticles promote DNA damage which is enhanced by chitosan thus suggesting that chitosan favors the nanoparticles’ internalization by cells and modulates their biological activity. The potential DNA damage should be considered – and potentially exploited for instance in anticancer treatment – when HA-based particles are used to devise biomaterials.