Aims Antibiotic resistance of different bacteria requires the development of alternative approaches for overcoming this phenomenon. The antibacterial effects of iron oxide (Fe3O4) nanoparticles (NPs) (from 50 to 250 mu g ml(-1)) on Escherichia coli antibiotic-resistant strains have been aimed. Methods and Results The study was performed with ampicillin-resistant E. coli DH5 alpha-pUC18 and kanamycin-resistant E. coli pARG-25 stains. Specific growth rate of bacteria (mu), lag phase duration and colony-forming units (CFU) were determined to evaluate growth properties. Fe3O4 NPs (average size of 10 center dot 64 +/- 4 center dot 73 nm) coated with oleic acid and synthesized by modified co-precipitation method were used. The medium pH, H+ efflux, membrane H+ conductance, redox potential determinations and H-2 yield assay were done using potentiometer methods. Growth properties were changed by NPs in concentration-dependent manner. NPs decreased (up to twofold) H+-fluxes through bacterial membrane more in E. coli in the presence of the N,N '-dicyclohexylcarbodiimide, inhibitor of ATPase, indicating that antibacterial activity of these NPs was connected with ATP-associated metabolism. Membrane-associated H-2 production was lowered up to twofold. Moreover, the synergetic interactions of NPs and antibiotics were found: combination of NPs and antibiotics provided the higher H+ conductance, lower H+-fluxes and H-2 yield. Conclusions Fe3O4 NPs can be suggested as alternative antibacterial agents, which can substitute antibiotics in different applications. Significance and Impact of the Study The antibacterial effects of Fe3O4 NPs on the growth properties and membrane activity of E. coli antibiotic-resistant strains have been demonstrated. These NPs have potential as antibacterial agents, which can substitute for antibiotics in bacterial disease treatment in biomedicine, pharmaceutical and environmental applications.