Superparamagnetic iron oxide nanoparticles (SPIONPs) are successfully synthesized in this study by co-precipitation method using actinobacterial metabolites as reducing agent. Physicochemical and morphological features of the nanoparticles (NPs) are analyzed by Fourier-transform infrared spectroscopy, x-ray-based techniques, vibrating sample magnetometer, thermal gravimetric analysis, and electron microscopic analysis, with an average size of 15–30 nm. Anticancer activity of the magnetite-NPs is systematically evaluated on HeLa cells using MTT assay, Hoechst nuclear staining, acridine orange/ethidium bromide dual staining and flow cytometric analysis. The obtained results open a new route for biosynthesis of SPIONPs, which to be used for various biomedical applications, particularly in cancer therapy.