Magnetite (Fe3O4) nanoparticles were prepared using coprecipitation and subsequently surface-functionalized with 3-aminopropyltriethoxysilane (APTS), polyethylene glycol (PEG), and tetraethoxysilane (TEOS). Nanoparticle morphology was characterized using scanning electron microscopy, while structure and stability were assessed through infrared spectroscopy and zeta potential, respectively. Average size of the nanoparticles analysed by dynamic light scattering was 89 nm, 123 nm, 109 nm, and 130 nm for unmodified magnetite and APTS-, PEG-, and TEOS-modified magnetite nanoparticles, respectively. Biological effect was studied on two bacterial strains: Gram-negativeEscherichia coliCCM 3954 and Gram-positiveStaphylococcus aureusCCM 3953. Most of modified magnetite nanoparticles had a significant effect onS. aureusand not onE. coli, whereas PEG-magnetite nanoparticles displayed no significant effect on the growth rate of either bacteria.