Biodegradation is a natural process, where the degradation of a xenobiotic chemical or pesticide by an organism is primarily a strategy for their survival. Pesticides are degraded in the environment principally by the action of microorganisms. Evaluate the ability of three bacterialstrains to chlorpyrifos-degrade, determine the chlorpyrifos degrading genes in the isolates, study their biochemical mode of degradation. Three bacterial strains i.e., Pseudomonas fluorescens, Rhizobium leguminosarum, and Bacillus megaterium were detected in Luria-Bertani (LB) medium degrading Chlorpyrifos at day 14. Microbiological, biochemical studies and genetic factors in biodegradation were studied. P. fluorescens had the highest efficiency with 58.90%, then R. leguminosarum 56.72%, and finally B. megaterium with 50.69%. According to the genetic similarity for degradation genes, B. megaterium and P. fluorescens have nearly the same, while R. leguminosarum has another pathway. P. florescence and R. Leguminosarum strains were the highest in the degradation process which analyzed the Chlorpyrifos component to 3,5,6-trichloro-2-pyridinol (TCP) and Chlorpyrifos Oxon that has a little toxic effect on the ecosystem. With the obtained results, it is recommended to apply these strains on soil and plants to reduce the toxicity of chlorpyrifos in the environment.