The aim of this study was to evaluate the acute effect of high-dose acetylsalicylic acid (ASA) on kidney and testis, and the potential protective and therapeutic effects of melatonin on ASA-related pathology. A total of 40 rats were randomly divided into the following 5 groups (n = 8): group 1: control, not given any drug; group 2: only 200 mg/kg ASA was given; group 3: 5 mg/kg melatonin was given 45 min before administering 200 mg/kg ASA; group 4: 5 mg/kg melatonin was given 45 min after administering 200 mg/kg ASA; and group 5: only 5 mg/kg melatonin was given. The histopathological changes and the biochemical findings; such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), reduced glutathione (GSH), and blood urea nitrogen (BUN) as well as serum creatinine (Cr) levels were evaluated. ASA significantly increased MDA levels in both kidney and testis, whereas it significantly decreased the values of SOD, CAT, GPX, and GSH in kidney and CAT levels in testis. Melatonin significantly decreased MDA levels in kidney and ameliorated it in testis, whereas it caused elevation in the levels of antioxidants. BUN and Cr levels were higher after ASA, whereas these levels were diminished after melatonin administration. The improvement obtained by melatonin on ASA-induced histological alterations was more prominent when it was used after ASA in kidney and before ASA in testis. In this study, we demonstrated the beneficial effect of melatonin on high-dose ASA-related pathology of kidney and testis for the first time.