Magnetic FeO nanoparticles (MNPs) functionalized with (3-aminopropylo)trietoksysilan (APTES) or N-carboxymethylchitosan (CMC) were proposed as nanocarriers of methotrexate (MTX) to target ovarian cancer cell lines. The successful functionalization of the obtained nanostructures was confirmed by FT-IR spectroscopy. The nanoparticles were characterized by transmission electron spectroscopy (TEM) and dynamic light scattering (DLS) techniques. Their potential zeta, magnetization, and hyperthermic properties were also explored. MTX was conjugated with the nanocarriers by ionic bonds or by amide bonds. The drug release kinetics were examined at different pH and temperatures. The MTT assay showed no toxicity of the MNPs[APTES] and MNPs[CMC]. Finally, the cytotoxicity of the nanostructures with MTX attached towards the ovarian cancer cells was measured. The sensitivity and resistance to methotrexate was determined in simplistic 2D and spheroid 3D conditions. The cytotoxicity tests of the tested nanostructures showed similar values for inhibiting the proliferation of ovarian cancer cells as methotrexate in its free form. Conjugating MTX with nanoparticles allows the drug to be directed to the target site using an external magnetic field, reducing overall toxicity. Combining this approach with hyperthermia could enhance the therapeutic effect in vivo compared to free MTX, though further research on advanced 3D models is needed.