Nanoparticle metal oxides offer a wide variety of potential applications in medicine due to the unprecedented advances in nanobiotechnology research. In this work, the effect of zinc oxide (ZnO) nanoparticles prepared by mechano–chemical method on the antibacterial activity of different antibiotics was evaluated using disk diffusion method against Staphylococcus aureus and Escherichia coli. The average size of ZnO nanoparticles was between 20 nm and 45 nm. Although ZnO nanoparticles (500 μg/disk) decreased the antibacterial activity of amoxicillin, penicillin G, and nitrofurantoin in S. aureus, the antibacterial activity of ciprofloxacin increased in the presence of ZnO nanoparticles in both test strains. A total of 27% and 22% increase in inhibition zone areas was observed for ciprofloxacin in the presence of ZnO nanoparticles in S. aureus and E. coli, respectively. The enhancing effect of this nanomaterial on the antibacterial activity of ciprofloxacin was further investigated at three different contents (500, 1000, and 2000 μg/disk) against various clinical isolates of S. aureus and E. coli The enhancing effect of ZnO nanoparticles on the antibacterial activity of ciprofloxacin was concentration-dependent against all test strains. The most enhancing activities were observed in the contents of the 2000 μg/disk. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010.