Unilateral ureteral obstruction (UUO) causes severe renal tubulointerstitial fibrosis. Because of many pharmacologic properties of thymoquinone (TQ), in this study, the effects of TQ against kidney fibrosis and dysfunction were investigated in rats with UUO. Forty male Wistar rats were divided into five groups: Sham operated, UUO, and the animals with UUO treated with losartan, captopril, or TQ. Collagen IV and transforming growth factor (TGF)‐β1 expressions, interstitial fibrosis, histological changes, and kidney function were assessed. UUO markedly increased renal expression of TGF‐β1 and collagen I and induced interstitial fibrosis (p < .001). Losartan, captopril, or TQ significantly downregulated the expression of these fibrotic markers and interstitial fibrosis (p < .01–p < .001). In UUO group, serum levels of urea and creatinine and protein excretion rate significantly increased, but glomerular filtration rate (GFR) and urine osmolarity showed a significant decrease (p < .001–p < .05). Administration of captopril and TQ caused no significant change in serum urea and protein excretion rate. Unlike losartan and captopril, TQ caused no significant alteration in GFR compared with Day 1. Losartan caused significant increases in serum urea and creatinine but significant decrease in urine osmolarity. TQ could be regarded as a potent therapeutic agent for treatment of UUO‐induced kidney fibrosis and dysfunction.