The broad area of magnetic iron oxide nanoparticle (M-IONP) applications and their exclusive physico-chemical characteristics (superparamagnetic properties per se, solubility and stability in aqueous solutions, and high bioavailability in vivo) make these nanoparticles suitable candidates for biomedical uses. The most employed magnetic iron oxides in the biomedical field are magnetite and maghemite. Cancer represents a complex pathology that implies multiple mechanisms and signaling pathways, this complexity being responsible for the increased resistance to therapy and the lack of an effective curative treatment. A potential useful alternative was considered to be the use of magnetic iron nanoparticles. The M-IONPs proved to be effective as contrast agents in magnetic resonance imaging, as drug delivery carriers for different therapeutic agents, in magnetic cell separation assays, and are suitable to be engineered in terms of size, targeted delivery and substance release. Moreover, their in vivo administration was considered safe, and recent studies indicated their efficiency as anticancer agents. This chapter aims to furnish an overview regarding the physico-chemical properties of M-IONPs (mainly magnetite, maghemite and hematite), the synthesis methods and their in vitro biological impact on healthy and cancer cell lines, by describing their potential mechanism of action-enucleation, apoptosis or other mechanisms.