Magnetic nanoparticles (NPs) have great potential for applications not only as catalysts or energy storage devices, but also in biomedicine, as contrast enhancement agents for magnetic resonance imaging, or for drug delivery. The same characteristics that make cobalt-based NPs so attractive raise serious questions about their safety. In this context, we investigated Co3O4-NPs. Believing that the characterization of NPs is relevant for understanding their biological activity, we analyzed them by atomic force and electron microscopy to define size, shape, and aggregation. To clarify whether their biological effects could be due to a potential release of cobalt ions, we evaluated spontaneous dissolution in different media. To determine their potential toxicity to human cells, we measured cell viability and ROS formation in two human cell lines using CoCl2 for comparison. Co3O(4)-NPs induced a concentration- and time-dependent impairment of cellular viability, although cobalt ions were more toxic. We also demonstrated that cobalt causes a rapid induction of ROS if supplied in the form of Co3O4-NPs rather than as ions. Moreover, we evaluated the cellular uptake of NPs. Interestingly, Co3O4-NPs are able to enter the cell very rapidly, remaining confined in vesicles inside the cytoplasm. They were found also inside the cell nuclei, though less frequently.