Iron oxide nanoparticles (FeO NPs) stabilized with Moringa oleifera (M.O.) were successfully synthesized. The study aimed to explore the cytotoxic, anti-proliferative and anti-microbial potential of FeO NPs through various assays, including trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), wound healing and disc diffusion assay. The Moringa oleifera-mediated FeO NPs (M O FeO NPs) underwent analysis using various techniques, including Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), UV-vis spectroscopy (UV-vis), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). FTIR, XRD and SEM-EDX results confirmed the successful synthesis of FeO NPs. The UV-Vis spectra analysis indicated an absorption peak at 314 nm, ensuring both the successful synthesis and remarkable stability of the nanoparticles. The nanoparticles displayed a uniform spherical morphology and contained Fe, O, confirming the formation of M O FeO NPs. Cytotoxic and anti-proliferative potential on MDA-MB 231 human breast cancer cells were observed with various concentrations of M O FeO NPs and the cytotoxicity result revealed an IC of 69.7μg/mL. In conclusion, stable FeO NPs were synthesized using a methanolic extract of M.O. and they demonstrated antimicrobial activity against both gram-negative and gram-positive bacteria. The nanoparticles exhibited cytotoxicity and anti-proliferative activity on MDA-MB 231 human breast cancer cell lines.