Antibacterial activity of Fe3O4 nanoparticles was investigated in three microorganisms including P. aeruginosa, E. Coli and Staphylococcus aureus. Fe3O4 nanoparticles were synthesized by chemical precipitation method (20 nm). The Fe3O4 nanoparticles antibacterial effect against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were studied by the culture method. First of all, each bacterium was grown in nutrient broth culture environment aerobically for 24 h at 37°C. After that, about 1 mL of each bacterium was placed onto a plate which was previously prepared by Mueller Hinton Agar culture environment. Their characteristics and physical properties were evaluated by X-ray diffraction (XRD), Alternating Gradiant Force Magnetometer (AGFM) and Transmission Electron Microscope (TEM). Antibacterial effects of Fe3O4 nanoparticles (1.56 to 25 mg/mL concentrations) against three pathogenic bacteria including Pseudomonas aeroginosa, Escherichia coli and Staphylococcus aureus were studied using well diffusion bacteriological test. The inhibition zone diameter was directly and strongly correlated to the nanoparticles concentration and Pseudomonas aeroginosa was the most sensitive bacterium to the Fe3O4 nanoparticles. The maximum diameter of inhibition zone was occurred for P. aeroginosa bacteria for both Fe3O4 samples. The most sensitive bacterium among three pathogenic bacteria tested was P. aeroginosa.