Malathion was used in vivo on fresh water fish Labeo rohita to study its toxicity. The acute toxicity tests were conducted during certain intervals in various concentrations (5, 10, 15, 20, 25 and 30 mg/L) of malathion. The physical and chemical analyses of water were carried out by following APHA methods. While treating with malathion, the percentage of fish mortality was assessed during 24, 48, 72 and 96 hours. The lethal and sub-lethal concentration of malathion were found to be LC₁₀₀ (25 mg/L) and LC₀ (5 mg/L), respectively. The antioxidant enzyme activity (Catalase 43.1±2.3, 16.5±0.57, 23.9±0.17 m moles of phenol liberated/min/100 mg protein and Glutathion-S-transferase (GST) 270.5±0.16, 143.2±1.03, 215.5±0.72 m moles of phenol liberated/min/100 mg protein), in the liver, muscle and gill, respectively increased during the accumulation of malathion, whereas it decreased (Catalase 17±1.44, 7.9±0.23, 10.7±0.69 m moles of phenol liberated/min/100 mg protein and GST 219.5±1.12, 108.1±0.34, 160.2±0.46 m moles of phenol liberated/min/100 mg protein) in the liver, muscle and gill respectively during depuration period. The effects of malathion resulted in the gradual decrease of nucleic acids, protein, free amino acids (FAA) and glycogen. During recovery period, the levels of biochemical components progressively increased indicating a probable recovery from the disruption of internal organ. Hence, the pesticide intoxication has made defective consequences in the normal metabolic pathways which led increasing the rate of morality in fish population.