New properties of nano-materials have made nanotechnology the leading part of biology and medical sciences. Due to their various biomedical properties, iron-based magnetic nanoparticles (MNPs) have been highly considered by biological researchers. Nowadays, increasing resistance to antibiotics is a major problem in treating clinical infections. Finding new antibacterial agents is therefore essential for the treatment of resistant strains. In this study, the iron oxide MNPs were produced using culture-medium supernatant of a newly isolated bacterium to investigate the inhibitory effects of the NPs on strains with a major role in clinical infections. Biosynthesis of iron oxide MNPs were detected by UV-Vis spectroscopy and the average size of particles was estimated by dynamic light scattering technique. The anti-bacterial activity of these NPs against E. coli and S. aureus was investigated using methods for the calculation of bacterial sensitivity coefficient. In the presence of NPs, the highest sensitivity coefficient value was observed for E. coli in 1xMIC concentration. On the other hand, S. aureus showed the lowest value. The death rate of the two strains in contact with NPs followed the first order kinetic equation and the survival rate decreased with the increase of exposure time. The results of this study as well as the high functionality of iron oxide MNPs, make its application desirable in the prevention and treatment of clinical infections.