This study aimed to investigate whether hesperidin and chrysin antioxidants have protective effects on renal injury induced by colistin in rats. Renal lipid peroxidation, total glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) enzyme activities, serum urea and creatinine levels, as well as tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) levels were determined. Injuries to the proximal and distal tubules were determined using histopathological and double immunohistochemistry examinations. The results showed that hesperidin and chrysin significantly decreased the levels of MDA and inflammatory parameters and significantly increased GSH, SOD, CAT, and GSH-Px levels against colistin-induced renal injury. The results also showed that cystatin C and calbindin D28K immunopositivities significantly increased through hesperidin and chrysin treatment. Hesperidin and chrysin alleviated the renal injury induced by colistin via anti-oxidant and anti-inflammatory activities. Thus, hesperidin and chrysin could attenuate colistin-induced nephrotoxicity via antioxidant and anti-inflammatory activities. Addition of hesperidin or chrysin to the treatment protocol of colistin treatment might benefit patient treatment in terms of the prevention of colistin-induced renal injury.