Acetaminophen (APAP) overdose is a major cause of acute hepatic and renal injury, primarily driven by excessive formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), glutathione depletion, and oxidative/nitrosative stress. Opuntia ficus-indica (L.) Mill. (OFI) fruit contains antioxidant and anti-inflammatory phytochemicals that may protect against xenobiotic-induced organ damage. This study investigated the hepatoprotective and nephroprotective effects of an aqueous OFI fruit extract against APAP-induced toxicity in rats, with emphasis on oxidative stress mechanisms. Adult male rats were pre-treated with OFI extract (100, 200, or 400 mg/kg, orally) for seven days, followed by a single APAP dose (3 g/kg) on day eight. After 24 h, serum biomarkers of hepatic and renal function (ALT, AST, ALP, total bilirubin, total protein, CREA, BUN, Na⁺, K⁺) and tissue oxidative stress indices (CAT, SOD, NO, and MDA) were evaluated. APAP administration caused substantial elevations in serum ALT, AST, ALP, CREA, and BUN, together with decreased antioxidant enzyme activities and increased lipid peroxidation in liver and kidney tissues. Pre-treatment with OFI at 100 and 200 mg/kg markedly ameliorated these alterations, improving biochemical markers and restoring antioxidant defence. In contrast, the 400 mg/kg dose failed to provide significant protection and exacerbated some parameters, indicating a biphasic, dose-dependent response. These findings demonstrate that low and moderate doses of OFI fruit aqueous extract exert significant hepatoprotective and nephroprotective effects against APAP-induced oxidative injury, likely through enhancement of antioxidant defence and attenuation of oxidative and nitrosative stress.