It has been reported that iron oxide nanoparticles have various biomedical applications, including cancer diagnosis and treatment. Iron oxide nanoparticles were known to exert cytotoxicity against MCF-7 breast cancer cell lines and in this present study, we have investigated for their apoptosis-inducing potential in the same cell line. The flow cytometry analysis of the MCF-7 breast cancer cell lines treated with functionalized iron oxide nanoparticles showed an increased percentage of cells in terms of viable, early, and late apoptosis. The cell cycle analysis of the MCF-7 cell lines treated with Syzygium aromaticum extract coated with polyvinylpyrrolidone (PVP) + iron oxide nanoparticles and PVP + iron oxide nanoparticles showed substantial accumulation of nanoparticles in the sub-G1 phase, confirming induction of apoptosis. The activities of caspase-3, caspase-8, and caspase-9 increased with increasing concentration of the nanoparticles indicating that activities of caspase can be activated by iron nanoparticles. Further, functionalized nanoparticles induced oxidative stress through reactive oxygen species (ROS) formation. Therefore, it is concluded that the functionalized iron nanoparticles induce apoptosis in MCF-7 breast cancer cell lines and further provides an opportunity to explore the iron nanoparticles for apoptosis in cancer treatment.