The aim of the present study was to investigate the effectiveness of melatonin and genistein in preventing radiation therapy (RT)-induced liver injury in mice. A total of 70 Swiss Albino male mice were divided into 7 equal groups (n=10/group) as follows: Melatonin (M group, G3), genistein (G group, G4), polyethylene glycol-400 (P group, G5), RT only (RT group, G2) and sham irradiation (C group, G1). RT plus genistein (RT+G group, G7) and RT plus melatonin (RT+M group, G6) were the co-treatment groups. Firstly, hepatic tissue damage was induced in mice via exposure to a single dose of 6-Gy irradiation. RT was performed with a cobalt-60 teletherapy machine (80 cm fixed source-to-surface distance, 2.5-cm depth). Melatonin was processed (100 mg/kg, intraperitoneal) 30 min before and genistein was administered (200 mg/kg, SC) one day prior to the single dose of irradiation. Six months following irradiation, all mice were sacrificed. The degree of liver injury was measured using histological liver sections. Liver injury was significantly worse in the RT group than in the control group (C; RT vs. C; P<0.05); however, liver injury decreased following co-treatment with melatonin or genistein vs. RT alone (RT+M and RT+G vs. RT; P<0.05). No difference was observed between the RT+M and RT+G groups (P>0.05). The present study revealed that melatonin and genistein administration prior to irradiation protects mice against liver injury, which may have therapeutic implications for RT-induced injuries.