In the present study, ultra-small, magnetic, oleyl amine-coated FeO nanoparticles were synthesized and stabilized with a cationic ligand, cetyltrimethylammonium bromide, and an anticancer drug, methotrexate, was incorporated into a micelle-like nanoparticle structure for glioblastoma treatment. Nanoparticles were further characterized for their physicochemical properties using spectroscopic methods. Drug incorporation efficiency, drug loading, and drug release profile of the nanoparticles were investigated. According to the results, max incorporation efficiency% of 89.5 was found for 25 µg/mL of methotrexate-loaded nanoparticles. The cumulative amount of methotrexate released reached 40% at physiological pH and 85% at a pH of 5.0 up to 12 h. The toxicity and anticancer efficacy of the nanoparticles were also studied on U87 cancer and L929 cells. IC concentration of nanoparticles reduced cell viability to 49% in U87 and 72% in L929 cells. The cellular uptake of nanoparticles was found to be 1.92-fold higher in U87 than in L929 cells. The total apoptosis% in U87 cells was estimated to be ~10-fold higher than what was observed in the L929 cells. Nanoparticles also inhibited the cell motility and prevented the metastasis of U87 cell lines. Overall, designed nanoparticles are a promising controlled delivery system for methotrexate to the cancer cells to achieve better therapeutic outcomes.