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In the present study, we explored the antibacterial potential of ZnO–nanoparticles that were biomimetically synthesized employing conditioned culture supernatant of Staphylococcus aureus (S. aureus). The in situ synthesized ZnO–NPs were characterized employing various biophysical techniques including UV-vis spectroscopy, electron microscopy, DLS and XRD analysis etc. Electron microscopic studies revealed the disintegration of bacterial cell wall upon interaction with synthesized ZnO–NPs. Various employed tests provided evidence that synthesized ZnO–NPs effectively inhibited less susceptible Gram-positive and Gram-negative bacterial isolates. Moreover, the synthesized ZnO–NPs inhibited the biofilm synthesis of tested microbes in vitro. Finally, the biomimetically synthesized ZnO–NPs successfully treated the S. aureus mediated experimental skin infection in BALB/c mice as well.