Acetonitrile is a hazardous solvent that is used in different industrial processes. It is a pernicious to human and environmental health. In this study investigates biodegradation of acetonitrile by nitrile hydrolysing enzymes produced by Pseudomonas species isolated from soil from contaminated industrial site. Physical and chemical examination of soil samples revealed an array of trace elements, and a moderate alkalinity followed by morphological, biochemical, and 16S rRNA gene sequencing performed. Qualitative and quantitative analysis of nitrile metabolising activity using bromothymol blue based method was done. All the five isolated strains used nitriles exclusively as sole carbon and nitrogen source. Thus, all five Pseudomonas strains showed positive nitrilase enzyme activity, as conformed by qualitative screening. In quantitative screening Pseudomonas aeruginosa GPC1-C had the highest nitrilase activity by producing 1.6±0.02 U/mL of ammonia, furthermore the isolated strains are subjected to degradation of acetonitrile additionally the research included the optimization of degradation efficiency using immobilize the resting sells in agar-agar matrices. Pseudomonas aeruginosa GPC1-K demonstrated the highest degradation efficiency among the isolated strains at 72h, when both resting and immobilized cells were subjected to different concentrations of acetonitrile. Both resting and immobilised Pseudomonas species produced nitrilase enzymes, demonstrating increasing efficiency in degrading acetonitrile compounds. This enzyme activity offers a promising solution for environmental bioremediation and reduce pollution.