Magnetic fluid hyperthermia is a modern cancer treatment that selectively heats tumor tissues to destroy them without harming healthy tissues. The current study aimed to evaluate the cytotoxic effect of Fe₃O₄/chitosan nanocomposite hyperthermia on liver cancer both in vitro and in vivo. The nanocomposite was prepared using the co-precipitation technique; and its magnetic and optical properties were measured along with its Raman spectrum. Cell toxicity for Fe₃O₄/chitosan nanocomposite was conducted on a liver cancer cell line (HepG2) using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, and the cell viability was performed after exposure to Fe₃O₄/chitosan nanocomposite hyperthermia using trypan blue stain. For the in vivo experiments, 25 male BALB/c albino micewere randomly/equally allotted into five groups: the 1^st group was the non-tumor control group, the 2^nd group involved the tumor-bearing control mice, the 3^rd group involved the tumor-bearing mice received intramuscular injection of Fe₃O₄/chitosan (90 mg/kg, once/week for two weeks), the 4^th and 5^th groups involved tumor-bearing mice received intramuscular injection of Fe₃O₄/chitosan and exposed to an alternating magnetic field with a frequency of "200 kHz" and an output current of "300 A" once or twice/week, respectively, for two weeks. The results showed that nanocomposite was able to induce cytotoxicity (in vitro), as well as enhanced programmed cell death and necrosis of tumor cells, and reduced significantly (P<0.05) the tumor size (in vivo), when exposed to a hyperthermal magnetic field. In conclusion, Fe₃O₄/chitosan nanocomposite could be a promising therapeutic option for liver cancer through magnetic heating technology.