Efficacy of three different bacterial species for biodegradation of malathion and its secondary products have been investigated. The concentration range of malathion under investigation was 25–200 mg⋅ L− 1. It has been observed that Pseudomonas putida was found to be most efficient for degradation of malathion. The removal of malathion was 72% at its concentration of 125 mg⋅ L− 1. The optimum parameters were studied for all three bacterial species in batch mode. The average values of K s and μ m a x were obtained for all these species for degradation of malathion. Results indicate that P. putida has high degradation potential than Rhodoccocus rhodochrous and Sphingomonas sp. The degradation of P. putida was maximum at concentration of 125 mg⋅ L− 1, pH and temperature at 7±0.2, 80° C respectively. Metabolites were obtained using GCMS analysis.