Abstract Magnetic iron oxide nanoparticles (magnetite) (MNPs) were prepared using different organic and inorganic bases. Strong inorganic base (KOH) and organic bases (NH4OH and 1,4-diazabicyclo[2.2.2]octane (DABCO)) were used in the syntheses of the MNPs. The MNPs were characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR) and magnetization measurements. MNPs prepared with strong inorganic base yielded an average size of 100nm, whereas the average size of the MNPs prepared with the organic bases was 150nm. The main competitive phase for MNPs prepared with the strong inorganic and organic bases was maghemite; however, syntheses with KOH yielded a pure magnetite phase. The transfection study performed with the MNPs revealed that the highest transfection rate was obtained with the MNPs prepared with KOH (74%). The correlation between the magnetic parameters and the transfection ratio without transfection agents indicated that MNPs prepared with KOH were a better vector for possible applications of these MNPs in biomedicine. HeLa cells incubated with MNP-KOH at 10μg/mL for 24 and 48h exhibited a decrease in population in comparison with the control cells and it was presumably related to the toxicity of the MNPs. However, the cells incubated with MNP-KOH at 50 and 100μg/mL presented a very small difference in the viability between the cell populations studied at 24 and 48h. These data illustrate the viability of HeLa cells treated with MNP-KOH and suggest the potential use of these MNPs in biomedical applications.