The biological synthesis of NPs using plant extracts plays an important role in the field of nanotechnology. In this study, magnetite nanoparticles (Fe3O4 NPs) were synthesized using a rapid, single step and completely green biosynthetic method by reducing ferric chloride hexahydrate and ferrous chloride tetrahydrate solution with Persea Americana leaf aqueous extract containing flavonoids and phenolic compounds as the main factor which acts as the reducing agent and efficient stabilizer. The structural and properties of the Fe3O4 NPs were investigated by UV-visible spectroscopy, fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM),transmission electron microscopy (TEM) and vibrating sample magnetometry (VSM). Moreover, the antimicrobial properties of Fe3O4 NPs were confirmed using the disk diffusion test and minimum inhibitory concentration and minimum bactericidal concentration on Staphylococcus aureus and Escherichia coli. The obtained results of disk diffusion test indicated that Fe3O4 NPs prevented the bacterial growth. Additionally, the obtained results demonstrated that the minimum inhibitory concentration and minimum bactericidal concentration of Fe3O4 NPs for Staphylococcus aureus were 12.5 mg/mLandfor Escherichia coli were 6.25 mg/mL. The findings of this study showed that Fe3O4 NPs can be used to inhibit the mentioned bacteria.