The aim of the present study was to compare the effects of melatonin and genistein on radiation-induced nephrotoxicity (RIN). A total of 70 Swiss Albino mice were divided into 7 groups. Five control groups were defined, which were sham irradiation (C, G1), radiation therapy only (RT, G2), melatonin (M, G3), genistein (G, G4) and polyethylene glycol-400 (G5), respectively. The co-treatment groups were the RT plus melatonin (RT+M, G6) and RT plus genistein (RT+G, G7) groups. Irradiation was applied using a cobalt-60 teletherapy machine (80-cm fixed source-to-surface distance, 2.5-cm depth). Melatonin was administered (100 mg/kg, intraperitoneal injection) 30 min before the single dose of irradiation, whereas genistein was administered (200 mg/kg, subcutaneous injection) 1 day before the single dose of irradiation. All the mice were sacrificed 6 months after irradiation. As an end point, the extent of renal tubular atrophy for each mouse was quantified with image analysis of histological sections of the kidney. Tissue malondialdehyde (MDA) levels were also measured in each animal. In the histopathological examination of the mouse kidneys, there was a statistically significant reduction (P<0.05) in the presence of tubular atrophy between the RT+M and RT+G groups and the RT group. There was a statistically significant increase in MDA levels in the irradiated versus sham groups (RT vs. C; P<0.05); however, MDA levels were significantly decreased by co-treatment with melatonin or genistein vs. RT alone (RT+M and RT+G vs. RT; P<0.05). In conclusion, the present experimental study showed that melatonin and genistein supplementation prior to irradiation-protected mice against RIN, which may have therapeutic implications for radiation-induced injuries.