Objective: Synthesis, characterization and antibacterial activity of iron oxide nanoparticles prepared by of Passiflora foetida aqueous extract. Methods: Iron Oxide nanoparticles (Fe2O3 NPs) were synthesized by the reduction of iron chlorides using phyto-chemicals as bio-reductants. The synthesis of Fe2O3 nanoparticles was confirmed by UV-Vis spectrophotometry and the average particle size was determined by X-ray diffractometry (XRD). Morphology of the synthesized Fe2O3 NPs was identified using Scanning Electron Microscopy (SEM). Further characterization of Fe2O3 NPs was done to study its nature by FTIR. Biological activity of Fe2O3 nanoparticles was analyzed through its antibacterial effect. Results: Synthesized iron oxide nanoparticles exhibit cubic crystalline phase. Morphology was identified as spherical and particle size ranges from 10 to 16 nm in diameter with an average size of 20 nm analyzed from XRD. Fe2O3 nanoparticles showed antibacterial activity as that of control drug (streptomycin) towards Gram positive and negative bacteria. Iron oxide nanoparticles were also found to be non-toxic as synthesized by using green synthesis. Conclusion: Green synthesis of Fe2O3 nanoparticles using Passiflora foetida leaves aqueous extract was considered to be relatively safe and particles possesses significant biological activity.