Recent investigations have attempted to understand and exploit the impact of magnetic field-actuated internalized magnetic nanoparticles (MNPs) on the proliferation rate of cancer cells. Due to the complexity of the parameters governing magnetic field-exposure though, individual studies to date have raised contradictory results. In our approach we performed a comparative analysis of key parameters related to the cell exposure of cancer cells to magnetic field-actuated MNPs, and to the magnetic field, in order to better understand the factors affecting cellular responses to magnetic field-stimulated MNPs. We used magnetite MNPs with a hydrodynamic diameter of 100 nm and studied the proliferation rate of MNPs-treated versus untreated HT29 human colon cancer cells, exposed to either static or alternating low frequency magnetic fields with varying intensity (40–200 mT), frequency (0–8 Hz) and field gradient. All three parameters, field intensity, frequency, and field gradient affected the growth rate of cells, with or without internalized MNPs, as compared to control MNPs-untreated and magnetic field-untreated cells. We observed that the growth inhibitory effects induced by static and rotating magnetic fields were enhanced by pre-treating the cells with MNPs, while the growth promoting effects observed in alternating field-treated cells were weakened by MNPs. Compared to static, rotating magnetic fields of the same intensity induced a similar extend of cell growth inhibition, while alternating fields of varying intensity (70 or 100 mT) and frequency (0, 4 or 8 Hz) induced cell proliferation in a frequency-dependent manner. These results, highlighting the diverse effects of mode, intensity, and frequency of the magnetic field on cell growth, indicate that consistent and reproducible results can be achieved by controlling the complexity of the exposure of biological samples to MNPs and external magnetic fields, through monitoring crucial experimental parameters. We demonstrate that further research focusing on the accurate manipulation of the aforementioned magnetic field exposure parameters could lead to the development of successful non-invasive therapeutic anticancer approaches.