Gentamicin, one of aminoglycosides, was greatly used in treatment of different bacterial infections in animal. This study investigated the hepato-nephrotoxicity induced by single intramuscular (IM) injection gentamicin in chickens. A total number of 90, one day-old, Balady chicks were randomly divided into 3 groups each with 30 birds. Group (1) served as a control; Group (2) was given 25 mg gentamicin /kg body weight (BW), Group (3) was given 50 mg gentamicin /kg BW. Hepato-nephrotoxicity was evaluated by measuring growth performances, mortality rats, hematological parameters, biochemical markers; reduced glutathione (GSH), nitric oxide (NO), liver function tests, serum urea and creatinine levels, as well as, by evaluation of histopathological pictures of liver and kidney. 50 mg gentamicin /kg BW reduced growth performance and decreased red blood cell (RBC) count, hemoglobin (Hb) concentration, mean corpuscular hemoglobin concentration (MCHC), total protein, albumin, globulin levels and albumin/ globulin (A/G) ratio. There were significant heterophilic leukocytosis and eosinophilia. Levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), urea, creatinine, liver and kidney NO were increased, meanwhile levels of GSH were decreased. Severe congestion, hemorrhage and multifocal coagulative necrotic areas were microscopically detected in liver and kidneys.Gentamicin injection altered hematological, biochemical and serum antioxidants pictures and induced hepatic and renal lesions in a dose dependent manner. The chickens administered 50 mg/kg BW gentamicin were more severely affected than 25 mg/kg BW gentamicin