Abstract: Background and Objective: The wide and indiscriminate use of pesticides for pest control in agriculture has inflicted serious harm and problems to humans as well as to the biodiversity. Microbial biodegradation of pesticides in contaminated soils has been considered advantageous to decontaminate areas that have been polluted by pesticides. The aim of this study consisted in isolation and molecular identification of potent bacterial isolates in pesticides biodegradation. Materials and Methods: The PCR amplification of 16sr DNA and GroEl genes were used to identify the potent bacterial isolates. The ability of bacterial isolates in pesticides biodegradation by gas chromatography was investigated. Detection and sequencing of oph gene in the bacterial isolates were employed. The enzymatically- generated degradation products of pesticides by GC/MS were detected. Results: Organophosphorus pesticides, diazinon and chlorpyrifos were the most persistent pesticides in the Egyptian soils samples. Four bacterial isolates GH10, GH2NO8, GH9OP and GH4SNO/P was identified as Cronobacter muytjensii strain GH10, Pseudomonas aeruginosa strain GH2NO8, Achromobacter xylosoxidans strain GH9OP and Pseudomonas putidas train GH4SNO/P, respectively based on 16s rDNA and GroEl gene sequence analysis. They were able to degrade 92.9, 91.82, 97.75 and 90.78% of diazinon at initial concentration (600 mg L–1) as compared to 16.99% in control and 93.43, 78.51, 93.18 and 95.36% biodegradation of chlorpyrifos at initial concentration (480 mg L–1) as compared to 4.28% in control, respectively, after 20 days. New organophosphorus hydrolase (oph) gene responsible for organophosphorus pesticides biodegradation was detected and sequenced in Cronobacter muytjensii strain GH10, Pseudomonas aeruginosa strain GH2NO8, Achromobacter xylosoxidans strain GH9OP, these sequences were deposited in GenBank under the accession number MF443872, MF443870 and MF443871, respectively. 3,5,6-trichloro-2-pyridinol (TCP) as metabolite of chlorpyrifos degradation and isopropyl-4-methyl-6- hydroxypyrimidine (IMHP) as metabolite of diazinon which were further metabolized to unknown polar metabolites was detected by GC/MS after 20 days in case of Cronobacter muytjensii strain GH10. Conclusion: The results of this study indicate that bacterial biodegradation is advantageous approach for pesticides bioremediation.