In the present study, silver, cadmium, iron and copper nanoparticles were biosynthesized by Salmonella typhimurium, Staphylococcus aureus, Candida albicans and Fusarium avenaceum strains. Analysis of the synthesized nanoparticles using the dynamic light scattering (DLS) revealed that the smallest nanoparticles were obtained by Salmonella typhimurium. The formation of the synthesized bio-AgNPs was confirmed by UV–Vis spectroscopy. The Ag, Cd, Fe and Cu nanoparticles produced by Salmonella typhimurium were separately immobilized on the chitosan (CS) framework by the impregnation method. The prepared CS-bio-nanocomposites were characterized by X-ray diffraction (XRD). The prepared compounds were investigated against three cancer cells (MCF-7, HeLa and HCT-1l6) for anticancer activity as well as against different microbial strains as antimicrobial activity. CS-bioAgNP composite clearly showed the greatest cytotoxicity against different cancer cell lines. Also, the CS-bioAgNP composite resulted in the highest inhibitory effect against different microbial growth strains using disk diffusion and kill time methods when compared with chitosan and other CS-bioNPs composites. Therefore, the present study provides evidence of novel antimicrobial and potent cytotoxicity characteristics of CS-bio-nanocomposite material that can be effectively utilized in the medical field.