Background: Acetaminophen (APAP) is the most widely used analgesic antipyretic drug, either by prescription or over the counter. APAP overdose can damage multiple organs, especially the liver and kidney, due to its toxic metabolites' formation and clearance. Aim: To investigate and compare the ameliorative effects of hesperidin (HSP) and melatonin (MEL) in rats with APAP-induced nephrotoxicity. Methodology: This comparative experimental study was carried out on 72 adult male Wistar albino rats were divided into nine groups (eight/each): groups (1-5): controls “negative, solvent, N-acetyl cysteine (NAC), MEL and HSP respectively; group (6): APAP-nephrotoxicity “rats were given a single dose (900 mg/kg) of APAP intraperitoneally (i.p.); group (7): APAP+NAC (150mg/Kg); group (8): APAP+MEL (10mg/Kg) and group (9): APAP+HSP (200mg/Kg). All treated drugs (NAC, MEL, and HSP) were given i.p. 30 minutes before APAP administration. Renal blood flow (RBF), blood urea, serum creatinine, tissue malondialdehyde (MDA), reduced glutathione (GSH), tumor necrotic factor alfa (TNF-α), and interleukin-6 (IL-6) were estimated by colorimetric methods. Immunohistochemical expression of inducible nitric oxide synthase (iNOS) was detected, and histopathological examination of renal tissue specimens was performed. Results: HSP, MEL, and NAC partially improved APAP-induced nephrotoxicity, evidenced by biochemical, histopathological, and immunohistochemical findings. Conclusion: Both HSP and MEL alleviated APAP-induced nephrotoxicity; however, HSP was superior to MEL, and it was as equal as NAC (which was also superior to MEL) as regards the tested parameters. The ameliorative effects of HSP and MEL may be at least in part due to their antioxidant and anti-inflammatory properties.