Biocidal activity and biocompatibility of nanomaterials (NMs) are crucial for healthcare applications. This study aims to develop biocidal hybrid NMs with high inhibition rates to control multidrug-resistant bacterial infection compared to conventional antibiotics. Herein, ZnO, chitosan-ZnO (CZnO) and alginate-ZnO (AZnO) NMs were synthesized via a simple one-pot technique. The one-pot process facilitates the efficiency of a chemical reaction whereby a reactant is subjected to successive chemical reactions in just one step. The resulted NMs bio-physicochemical features were analyzed using various analytical methods. The bactericidal and bacteriostatic mechanism of NMs strongly depends on the production of reactive oxygen species in NMs, due to their size, large surface areas, oxygen vacancies, ion release, and diffusion ability. The antibacterial potential of the NMs was tested against methicillin-resistant Staphylococcus aureus. The inhibition zone disclosed that the AZnO possessed an excellent antibacterial activity compared to ZnO and CZnO. Furthermore, toxicity studies revealed that the AZnO demonstrated low toxicity to the HepG2 cell lines. These results confirmed that the AZnO hybrid nanomaterials are promising futuristic biocidal agents suitable for the clinical and healthcare industries.