Chemical pesticides have an impact on other living organisms in addition to their intended target organisms. Any chemical pesticide is therefore made safe for use by examining its biological characteristics and side effects. The present study was aimed at determining the resistance efficiency of six bacterial isolates obtained from malathion-contaminated soils. Bacteria were isolated from soil samples collected in Adhamiya, Baghdad, Iraq. Biochemical tests and VITEK 2 compact equipment were used to identify the bacterial isolates. Primary and secondary screening tests were conducted on the bacterial isolates for resistance against malathion pesticides. The optimal bacterial growth conditions were determined in malathion-contaminated media. The results demonstrated that the bacterial isolates 1, 3, 4, and 8 grew best on malathion-contaminated (100 mgL-1) mineral salt medium (MSM). Isolates 1 and 2 had a MIC of 500 mgL-1, where they continued to grow until the seventh day of incubation. Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas hydrophilia, and Escherichia coli were the identified bacterial isolates. These isolates showed optimal bacterial growth in the test conditions (temperature, incubation period, and pH), indicating their efficiency and ability to degrade malathion. The highest average growth of P. aeruginosa at 35 °C was 70.08 mm, while that of P. putida was 40.10 mm during the 7-day incubation period. Also, the highest values of average growth were observed in the same bacteria at pH 7, with a value of 26.98 mm. The findings of this study reveal that Pseudomonas aeruginosa and Pseudomonas putida were the best bacterial isolates for biodegrading malathion.