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The incidence of female breast cancer has increased; it is the most commonly diagnosed cancer, at 11.7% of the total, and has the fourth highest cancer-related mortality. Magnetic nanoparticles have been used as carriers to improve selectivity and to decrease the side effects on healthy tissues in cancer treatment. Iron oxide (mainly magnetite, Fe3O4), which presents a low toxicity profile and superparamagnetic behavior, has attractive characteristics for this type of application in biological systems. In this article, synthesis and characterization of magnetite (NP-Fe3O4) and silica-coated magnetite (NP-Fe3O4/SiO2) nanoparticles, as well as their biocompatibility via cellular toxicity tests in terms of cell viability, are carefully investigated. MCF-7 cells, which are commonly applied as a model in cancer research, are used in order to define prognosis and treatment specifics at a molecular level. In addition, HaCaT cells (immortalized human keratinocytes) are tested, as they are normal, healthy cells that have been used extensively to study biocompatibility. The results provide insight into the applicability of these magnetic nanoparticles as a drug carrier system. The cytotoxicity of nanoparticles in breast adenocarcinoma (MCF-7) and HaCat cells was evaluated, and both nanoparticles, NP-Fe3O4/SiO2 and NP-Fe3O4, show high cell viability (non-cytotoxicity). After loading the anti-tumor drug doxorubicin (Dox) on NP-Fe3O4/Dox and NP-Fe3O4/SiO2/Dox, the cytotoxicity against MCF-7 cells increases in a dose-dependent and time-dependent manner at concentrations of 5 and 10 μg/mL. HaCat cells also show a decrease in cell viability; however, cytotoxicity was less than that found in the cancer cell line. This study shows the biocompatibility of NP-Fe3O4/SiO2 and NP-Fe3O4, highlighting the importance of silica coating on magnetic nanoparticles and reinforcing the possibility of their use as a drug carrier system against breast adenocarcinoma cells (MCF-7).