To enhance the removal efficiency of malathion in the wastewater from organophosphate pesticide mill, a bacterium, Acinetobacter johnsonii MA19, that could degrade malathion with cometabolism was isolated from malathion-polluted soil samples using enrichment culture techniques. Four kinds of additional compounds, sodium succinate, sodium acetate, glucose, and fructose were tested to choose a favorite carbon source for the cometabolism of strain MA19. The results showed that sodium succinate and sodium acetate could promote malathion biodegradation and cell growth. The investigation results of effects of sodium succinate concentrations on the malathion biodegradation indicated that the more sodium succinate supplied resulted in quick degradation ofmalathion and fast cells multiplied. Zero-order kinetic model was appropriate to describe the malathion biodegradation when the concentration of sodium succinate was more than 0.5144 g/L. The degradation rate constant (K) reached the maximum value of 3.5837 mg/(L·h) when the mass ratio of sodium succinate to malathion was 128.6 mg/mg. The aquatic toxicity of the malathion was evaluated using the test organism, Limnodrilus hoffmeisteri. The data obtained suggested that the toxicity of malathion could be ignored after 84 h biodegradation. Our result demonstrates the potential for using bacterium A. Johnsonii MA19 for malathion biodegradation and environmental bioremediation when some suitable conventional carbon sources are supplied.