In this study, iron oxide nanoparticles (Fe 3 O 4 NPs) were synthesized using a completely green biosynthetic method by reduction of ferric chloride (FeCl 3 ) solution with Mango leaves extract. The preparation of iron oxide nanoparticles (IONPs) through the biochemical reduction of 25 ml of 4g (0.12M) iron chloride ( III ) solution by 5 ml of 6 g Mango leaves extract to determine the optimum pH, time and both concentration of the substrate, and the precursor in order to obtain small particle size and size distribution which needed for biomedical applications. The iron oxide nanoparticles were successfully prepared at optimum pH=8, concentration of Fe 0.12M, substrate mass 6 g with 5:25 ratio (extract to precursor in ml). The synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and UV-visible spectroscopy. The UV-Visible spectrum of the colloidal solution has absorbance peaks observed at 293 nm. FTIR data reveal to identify the potential bio-molecules in the leaf extract responsible for the reduction and also the capping reagent responsible for the stability of the bio-reduced iron oxide nanoparticles. The structural properties of the Fe 3 -NPs were investigated by X-ray diffraction and their toxicities were evaluated by means of histological examinations against Escherichia coli (gram -ve) and Staphylococcus aureus (gram +ve). test organisms. Keywords: Iron oxide nanoparticles, green synthesis, and antibacterial activities O 4 3 +