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Green synthesis of nanoparticles (NPs) is a more sustainable, safe, and environmentally friendly method. This study aimed to investigate the synthesis procedure of iron oxide nanoparticles (IONPs) using the seed extract of Nigella sativa (N. sativa) as a strong reducing agent and to estimate their cytotoxic and antibacterial properties. The obtained IONPs were characterized by field-emission scanning electron microscopy, X-ray diffraction, dynamic light scattering (DLS), zeta potential (ZP), and Fourier-transform infrared spectroscopy analyses. The cytotoxicity of the biosynthesized IONPs was demonstrated by the MTT on a Vero cell line. Furthermore, the antibacterial activity of the fabricated biosynthesis metal oxide NPs was tested on Escherichia coli (E. coli) (ATCC 35218) and Staphylococcus aureus (S. aureus) (ATCC 29213) bacterial strains using the Kirby–Bauer disk diffusion method. This study showed the formation of a well-dispersed, highly stable (ZP ζ = −51.8 mV) IO NPs with an average diameter of about 31.45 nm. Moreover, the biosynthetic NPs (IONPs) exhibited a significantly noncytotoxic effect when analyzed by the MTT assay. The biosynthetic NPs (NS-IONPs) exhibited excellent antibacterial activity against E. coli and S. aureus, where the inhibition zones were 12.34 ± 0.58 and 11.52 ± 0.58, respectively.