Organophosphorus pesticides form a diverse group of chemicals, having a wide range of physicochemical properties with crucial toxicological actions and endpoints. These are extensively used to control pests of different food (fruits, vegetables, tea, etc.) and non-food (tobacco, cotton, etc.) crops. Malathion is an important widely used organophosphorus pesticide but its hepatotoxic effects on fish are not well studied. Therefore, the current study was designed to investigate the hepatotoxic effects of Malathion on rohu (Labeo rohita) fish in a semi-static system using different parameters. The LC50 of Malathion was found to be 5 µg/L for rohu for 96 h through Probit analysis and was used for further toxicity testing. To find the hepatotoxic effects of Malathion, changes in different biochemical indices including protein contents, Lipid Peroxidation (LPO), activities of four protein metabolic enzymes [Aspartate Aminotransferase (AAT), Lactate Dehydrogenase (LDH), Alanine Aminotransferase (AlAT), and Glutamate Dehydrogenase (GDH)], seven antioxidant enzymes [Catalase (CAT), Superoxide Dismutase (SOD), Peroxidase (POD), Glutathione (GSH), Glutathione Reductase (GR), Glutathione-s-transferase (GST), and Glutathione Peroxidase (GSH-Px)], DNA damage [in term of comet tail length, tail moment, DNA percentage in tail, and olive tail moment], reactive oxygen species (ROS), and Histopathological alterations were assayed. Malathion exposure led to a time-reliant significant (P < 0.05) decrease in protein contents and a significant (P < 0.05) increase in ROS, LPO, enzymatic activities, and DNA damage. The histopathological examination of the liver showed different changes including hepatic necrosis, fatty infiltration, hemorrhage vacuolation, glycogen vacuolation, congestion, and cellular swelling. The current study clearly revealed Malathion as a potent hepatotoxic pesticide; therefore the injudicious, indiscriminate and extensive use of Malathion should be prohibited or at least reduced and strictly monitored.