It is described the reproducible formulation and complete physicochemical characterization of nanohybrids based on magnetite (Fe3O4) cores embedded within a polyethylenimine (PEI) matrix. Particle size, surface electrical charge, X-ray diffraction and Fourier transform infrared spectroscopy (FTIR) analyses, and magnetic field-responsive behaviour characterizations defined that the 4:3 (Fe3O4:PEI) weight proportion led to the best production performances of magnetically responsive nanocomposites in which the magnetic nuclei are completely covered by the polymeric shell. Agarose gel electrophoresis assays demonstrated the capacity of the Fe3O4/PEI particles to condense, release, and protect the DNA against enzymatic degradation. In vitro assays were performed to evaluate the transfection efficiency (up to 4.5% of transfected HEK-293 cells at a 10/1 PEI/DNA ratio), iron absorption by D1-mesenchymal stem cells (D1-MSCs, high values after only 15 min of magnetic incubation), influence on metabolic activity (negligible effect up to 44 μg nanocomposites/105 cells), and cell isolation capacity of the core/shell particles (significant increase in the retention of D1-MSCs transduced with green fluorescent protein). The Fe3O4/PEI nanohybrids hold promising characteristics suggestive of their capacity for transfection and cell isolation applications.