Silver and magnetic (Fe3O4) nanoparticles have attracted wide attention as novel antimicrobial agents due to their unique chemical and physical properties. In order to study the comparative effects on antibacterial and animal cytotoxicity, Staphylococcus aureus and NIH 3T3 fibroblasts were used, respectively. Both nanoparticles were synthesized via a novel matrix-mediated method using poly(ethylene) glycol. Formation of silver nanoparticles was confirmed by fluorescence and ultraviolet-visible spectroscopic techniques. The poly(ethylene) glycol-coated silver and Fe3O4 nanoparticles were characterized by scanning electron microscope, transmission electron microscope, zeta potential, particle size analysis, Fourier-transform infrared, X-ray diffraction, and X-ray photoelectron spectroscopy. The antimicrobial results indicate that both poly(ethylene) glycol-coated silver and Fe3O4 nanoparticles inhibited S. aureus growth at the concentrations of 5 and 10 µg/mL at all time points without showing any significant cytotoxicity on NIH 3T3 fibroblasts. The particle size of both the poly(ethylene) glycol-coated silver and Fe3O4 nanoparticles dominated in the range 10-15 nm, obtained by particle size analyzer. The poly(ethylene) glycol coating on the particles showed less aggregation of nanoparticles, as observed by scanning electron microscope and transmission electron microscope. The overall obtained results indicated that these two nanoparticles were stable and could be used to develop a magnetized antimicrobial scaffolds for biomedical applications.