Nitrilase (EC 3.5.5.1) is an important biological catalyst with industrial application, which can directly convert nitrile compounds into corresponding carboxylic acids and ammonia under mild conditions. Nitrilase is used to convert molecules containing nitrile groups into carboxylic acid derivatives, and it is also used in many industries such as the textile industry. Studies should be conducted on the production of nitrilase, which is widely used in industry and has the potential to be used in other industrial areas, from different sources. Since reducing the cost in the industry will reflect positively on the consumer, high-activity nitrilases should be obtained. For this purpose, optimization of the production mediums gains importance in the production of nitrilase from microbial sources. The present study deals with the production and optimization of the microbial nitrilase (EC 3.5.5.1). Microbial strains stored in the laboratory were used in this study. All selected strains ( Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), Pseudomonas putida (ATCC 17514), Pseudomonas fluorescens (DSM 6521), Pseudomonas oleovorans (DSM 50188), and Micrococcus luteus (DSM 20030)) from stock were screened on 3 different mediums. The acetonitrile showed to be an inducer for nitrilase production. The most active bacterium produced about 41.11 U/ml of nitrilase activity and was identified as Pseudomonas putida (ATCC 17514). Maximum nitrilase production was obtained using the medium (3) with 0.50 g/L (v/v) acetonitrile as an inducer. The effects of different parameters (chemical and physical) on the nitrilase (EC 3.5.5.1) production were studied. When bacterial growth at pH 8.0 and 28 ºC was cultured for 20 h, and medium containing starch as a carbon source and peptone-yeast extract as a nitrogen source, the nitrilase activity peaked at 172.76 U/ml. Enzyme activity could be increased 4.2 times with optimization studies. By optimization of enzyme production, a significant increase in activity may be achieved. In this study, when compared with similar studies about nitrilase production from bacteria in the literature, high-activity nitrilase production from Pseudomonas putida (ATCC 17514) was achieved. Graphical Abstract