Our review summarizes the latest approaches regarding in vivo biocompatibility evaluation of magnetite nanoparticle-based systems. The paper follows the applications of Fe3O4 nanoparticles in cancer diagnosis and treatment, by means of nanoparticle-mediated magnetic hyperthermia, respectively by targeted delivery of chemotherapeutics. The long-term biodistribution in relevant organisms is also discussed, due to the need of knowing the exact course of magnetite nanoparticles after the fulfillment of their function. Several commercial Fe3O4 systems used as contrast agents for medical imaging and cancer treatment by hyperthermia are briefly presented in the last section.