The synthesis of nanosized Ag/Fe codoped hydroxyapatite (HAp) nanocomposite materials with antibacterial and anticancer characteristics is highly attractive for advancing the development of biological applications. The objective of this study was to evaluate the antibacterial and anticancer characteristics of Ag/Fe codoped hydroxyapatite materials. We developed a facile chemical precipitation method for the fabrication of Ag/Fe:HAp nanocomposites. The developed Ag/Fe:HAp nanocomposite materials were characterized with Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Raman spectroscopy, and transmission electron microscopy (TEM). For measuring the size of Ag/Fe:HAp nanocomposites, dynamic light scattering (DLS) is an advantageous method. The chemical states and chemical composition of Ag/Fe:HAp were observed by X-ray photoelectron spectroscopy (XPS) analysis. In addition, the antibacterial efficacy of Ag/Fe:HAps against Gram-positive (S.aureus), and Gram-negative (S.typhi, and E.Coli) microorganisms is examined in this current study. Ag/Fe:HAp nanocomposite materials have been evaluated for biological toxicity in vitro, and the results showed that the particles were excellent at identifying and killing cancer cells. In this respect, Ag/Fe:HAp nanocomposites significantly impact human colon cancer cells (HT29) while have no effect on normal fibroblast cells (L929).