High spin polarization and low resistivity of Fe3O4 at room temperature have been an appealing topic in spintronics with various promising applications. High-quality Fe3O4 thin films are a must to achieve the goals. In this report, Fe3O4 films on different substrates (SiO2/Si(100), MgO(100), and MgO/Ta/SiO2/Si(100)) were fabricated at room temperature with radio-frequency (RF) sputtering and annealed at 450 °C for 2 h. The morphological, structural, and magnetic properties of the deposited samples were characterized with atomic force microscopy, X-ray diffractometry, and vibrating sample magnetometry. The polycrystalline Fe3O4 film grown on MgO/Ta/SiO2/Si(100) presented very interesting morphology and structure characteristics. More importantly, changes in grain size and structure due to the effect of the MgO/Ta buffering layers have a strong impact on saturation magnetization and coercivity of Fe3O4 thin films compared to cases of no or just a single buffering layer.