<p>Iron oxide nanoparticles (IONPs) show promise in a range of biological applications. In this study, IONPs were synthesized from <i>Penicillium simplicissimum</i> (<i>P. simplicissimum</i>) and further capped with Bovine Serum Albumin (BSA) (capped-IONPs). The color change of the extract treated with ferric chloride (FeCl₃) indicates the formation of IONPs. These IONPs were further conjugated with BSA, and further observed in color change indicates that capping has taken place. Characterization process was carried out through various microscopic techniques such as UV-vis spectrophotometry, FTIR, EDX, SEM, TGA, TEM and Zeta Potential analysis. Characterization of capped-IONPs showed that UV-visible spectroscopy had a peak at 285 nm, confirming nanoparticle formation. FTIR detected amine and amide groups at 3299 cm-1 and 1636 cm-1. SEM showed the nanoparticles were spherical, and EDX confirmed the presence of iron. TEM revealed their size ranged from 3.42 to 5.41 nm. The zeta potential was +22.15 ± 0.21 mV, indicating good stability, and TGA confirmed their thermal stability. The capped-IONPs demonstrated potential toxicity towards HCT116 and HT29 colorectal cancer cells with IC₅₀ of 34 ± 1.5 µg/ml and 54 ± 1.0 µg/ml while less toxic towards colon healthy cells, CCD112CoN (IC₅₀ 168 ± 1.0 µg/ml), respectively using MTT assay after 24 h exposure. Capped-IONPs triggered apoptosis in colorectal cancer cells by generating reactive oxygen species (ROS), which disrupted the mitochondrial membrane potential (Δψm) and led to significant DNA fragmentation in HT29 and HCT116 cell lines compared to untreated cells. The treatment also elevated p53 levels in both cell lines, demonstrating the ability of capped-IONPs to induce apoptosis and inhibit cancer cell proliferation. These results showed that capped-IONPs significantly inhibit colorectal cancer growth by inducing apoptosis and can be used for nanodrug delivery to treat colorectal cancer.</p> Graphical abstract <p></p>