Background: The enzyme arginase plays a significant role in the growth of many organisms as important biological enzyme. Objective: The study was designed to isolate L-arginase from P. aeruginosa that isolated from sewages and to evaluate the influence of arginase on biofilm-generating bacteria. Materials and Methods: The current study includes 28(54.8%) isolates from soil with 24 (46.2%) from sewage water out of 52 isolated from soil and sewage for the purpose of obtaining Pseudomonas aeruginosa. Using standard laboratory methods to isolate Pseudomonas aeruginosa, 33 isolates were isolated, of which 15 (45.45%) were from soil and 18 (54.54%) from sewage. The study also included 177 different eyes (urinary fluid, excrement, blood, wounds, sputum, and cerebrospinal fluid) of patients suffering from various injuries. Results: The statistical analysis of the isolates indicated that there is a high significant (P≤0.01) for the growth of some types of bacteria, such as P. aeruginosa 30(16.89%), Klebsiella, Serratia 33(18.88%) and Acinetobacter 33(18.88%). While the results showed that Lactobacillus is the lowest number of isolates 7(3.9%). Optimum condition of arginase activity and stability comprising pH level of eight degrees, the temperature of 37℃ is the best conditions for the production of arginine, concentrations of L-arginine substrate (0.1-2.5)g/l on the activity of arginase enzyme for arginine production, The finding also shows that the arginase enzyme exhibited the highest activity after 96 hours of the incubation process L- arginase Purification Assay including Ammonium Sulphate precipitation in (60-80)%, Dialysis, Ion exchange chromatography, Gel filtration. Conclusions: This study concluded that the enzyme arginase had a significant effect on the strong biofilm of pathogenic bacteria.