Gentamicin (GNT) as an aminoglycoside antibiotic is commonly used against life threatening bacterial infections, however, the risk of nephrotoxicity is the main limitation of GNT therapeutic indication. Aim of the Work: This study was designed to evaluate the potential protective effects of apocynin (APO) and melatonin (MEL) against GNT induced nephrotoxicity in rats. Material and Methods: This study was carried out on thirty-two adult male albino rats randomly divided into 4 equal groups, GroupI (negative control), GroupII (GNT group) each rat was given intraperitoneal (ip) injection of (100mg/kg) of GNT daily for 7 days, Group III (APO/GNT) each rat was given (ip) injection of (10mg/kg) APO for 7 days started before administration of APO (10mg/kg) (ip) plus GNT (100mg/kg) (ip) for another 7 days. Group IV (MEL/GNT) each rat was given (ip) injection of (15mg/kg) MEL for 7 days started before administration of MEL (15mg/kg) (ip) plus GNT (100mg/kg) (ip) for another 7 days. Results: Gentamicin induced nephrotoxicity, as rats received GNT significantly presented an increase in the 24 -h urine volume, renal somatic index (RSI), urine protein, creatinine (Cr), blood urea nitrogen (BUN), serum lactate dehydrogenase (LDH), and malondialdehyde (MDA) Furthermore, GNT induced a significant decrease in the body weight gain percentage, creatinine clearance (CCr), superoxide dismutase (SOD) and glutathione peroxidase (GPX) in comparison to control rats. Gentamicin also induced hypercellularity in mesangial cells and renal tubular epithelium degeneration. Either APO or MEL significantly decreased 24- h urine volume, RSI, urine protein, Cr, BUN, , LDH, and MDA additionally, either APO or MEL caused a significant increase in body weight gain percentage, CCr, SOD, and GPX when compared to GNT group, moreover, either APO or MEL showed ren-oprotective effects histopathologically. Conclusion: Apocynin and melatonin can attenuate nephrotoxic effects in rats treated with GNT possibly through anti-oxidant effect and keeping normal renal tissue morphology.