Microbial antibiotics resistance is considered a serious health issue in the Middle East and developing countries. In this study, the Fe2O3 nanoparticles was prepared chemically, and the particles size and shape were analyzed by using Scan electron microscope (SEM) and X-Ray diffraction (XRD). Different concentration of Fe2O3 nanoparticles were used and examined on E.coli and S. aureus. Using liquid dilution and in vitro cytotoxicity assay by microplate toxicity test (MTT). The microbial cell metabolic activity was measured on gram-negative, gram-positive bacteria and fungi after treating with different concentrations of Fe2O3 nanoparticles. The results of liquid dilution method showed that the MIC of Fe2O3 nanoparticles are 30 μg/ml and 40 μg/ml on E.coli and S. aureus respectively. The results of MTT assay exhibited the ability of Fe2O3 nanoparticles to eliminate the gram negative bacteria (E.coli and K. pneumoniae) at 20 µg/ml, while S. aureus, M. luteus, Candida albicans and Candida parapsilosis were totally eliminated at 30 µg/ml.