The Fe₃O₄-PEG magnetic nanoparticles (NPs) were prepared by hydrothermal method at different concentrations (FeCl₃·6H₂O 0.75 mg/mL and FeCl₃·6H₂O 1.5 mg/mL) and subsequently surface-functionalized coating with polyethylene glycol (PEG), the successful coating of PEG molecules on the surface of Fe₃O₄. These magnetic NPs exhibited good dispersibility and dissolvability in physiological condition. The obtained magnetic nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG) and vibrating sample magnetometer (VSM). The antibacterial activity of Fe₃O₄-PEG magnetic nanoparticles (MNPs) was studied against two bacterial strains: Gram-positive Staphylococcus and Gram-negative Escherichia coli aureus. The modified MNPs had a significant effect is more on S. aureus and less on E. coli. The results showed that polyethylene glycol-functionalized magnetic (Fe₃O₄) NPs as a novel DNA-mediated antibacterial agent.