This research showed that the iron nanoparticles (FeNPs) formulated with Ocimum basilicum aqueous extract having potent antioxidant and anti-human glioma activities in vitro condition. For determinate the properties of the FeNPs that were produced from the reaction between iron chloride solution with aqueous Ocimum basilicum extract, we used UV–Visible Spectroscopy (UV-Vis), Field Emission Scanning Electron Microscopy (FE‐SEM), and Fourier Transformed Infrared Spectroscopy (FT‐IR). To survey the cytotoxicity and anti-human glioma effects of FeNPs, MTT assay was used on the SW1088, LN-229, U-87 and A-172 cell lines. For investigating the antioxidant properties of FeNPs, the DPPH test was used in the presence of butylated hydroxytoluene as the positive control. Iron nanoparticles had very low cell viability and anti-human glioma properties dose-dependently against SW1088, LN-229, U-87 and A-172 cell lines without any cytotoxicity on the normal cell line. The IC50 of the iron nanoparticles were 101, 75, 97, and 54 µg/ml against SW1088, LN-229, U-87 and A-172 cell lines. DPPH test revealed similar antioxidant potentials for FeNPs and butylated hydroxytoluene. It seems that the anti-human glioma effects of recent nanoparticles is due to their antioxidant properties.