An innovative biomimetic approach has been employed for the synthesis of human and environmental friendly nanomaterials for biomedical application. The aim of this research is to study the structural analysis of biomimetic-synthesized gold (Au) and silver (Ag) nanoparticles loaded on pristine hydroxyapatite (HAp) and their potential advances in biomedical applications. The synthesized metal hydroxyapatite nanoparticles' structural and morphological characteristics were studied using X-ray diffraction (XRD), different spectroscopies (UV-vis, DRS, FTIR, zeta potential), BET surface area and pore size analyzer and electron microscopy (FE-TEM, FE-SEM). Biological activity was established using osteoblast-like MG-63 cell line and antibacterial tests with Escherichia coil (E. coli), which is one of the most common bacteria in the human body. Cell cytotoxicity test (MTT assay and fluorescence imaging with AO/PI staining) was conducted with MG-63 cells to study the toxicity of Au-HAp, Ag-HAp, and Au-Ag-HAp (bimetallic) nanoparticles. Antimicrobial studies have shown that all Ag-HAp nano particles have excellent in vitro antibacterial activity with E. coli. The present research investigates the structural stability and biocompatibility of Au-HAp, Ag-HAp, and Au-Ag-HAp nanoparticles, which may be the best materials to repair bone infection (osteomyelitis) and could be useful as a potential scaffold material to prevent postoperative infections.