Nowadays, the influence of nanoparticles (NPs) on microorganisms attracts a great deal of attention as an alternative to antibiotics. Iron oxide (Fe3O4) NPs' effects on Gram-negative Escherichia coli BW 25113 and Gram-positive Enterococcus hirae ATCC 9790 growth and membrane-associated mechanisms have been investigated in this study. Growth specific rate of E. coli was decreased, indicating the bactericidal effect of Fe3O4 NPs. This inhibitory effect of NPs had a concentration-dependent manner. The reactive oxygen species together with superoxide radicals and singlet oxygen formed by Fe3O4 NPs could be the inhibition cause. Fe3O4 NPs showed opposite effects on E. hirae: the growth stimulation or inhibition was observed depending on NPs concentration used. Addition of NPs altered redox potential kinetics and inhibited H-2 yield in E. coli; no change in intracellular pH was determined. Fe3O4 NPs decreased H+-fluxes through bacterial membrane more in E. coli than in E. hirae even in the presence of DCCD and increased ATPase activity more in E. hirae than in E. coli. Our results showed that the Fe3O4 NPs demonstrate differentiating effects on Gram-negative and Gram-positive bacteria likely due to the differences in bacterial cell wall structure and metabolic peculiarities. Fe3O4 NPs of different concentrations have no hemolytic (cytotoxic) activity against erythrocytes. Therefore, they can be proposed as antibacterial agents in biomedicine, biotechnology, and pharmaceutics.