The aim of this study was to investigate and trace the biodegradation products of the pesticide malathion in a comparative manner by two different lactobacilli strains; L. casei (NRRL1922) and L. acidophilus (NRRL 23431). The two strains were cultivated separately into skimmed milk supplemented with 5 ng/ml malathion. After incubation under the appropriate conditions, randomized samples were taken at intervals 24, 48, 72 and 120 hours along with control samples and analyzed for the presence of malathion and its degradation products by the GC-MS spectrometry; As well as, analyzed to record the level of phosphatase enzyme which suggested to be involved in the biodegradation process. The results showed a high ability of the two tested strains to degrade malathion with a superiority of L. acidophilus (NRRL 23431) over L. casei (NRRL 1922). The level of phosphatase enzyme was elevated in both strains in the presence of malathion and decreased gradually upon the depletion of malathion from the sample, which reflects the role of the phosphatase enzyme in the biodegradation process.