Proteases, a class of enzymes, play a crucial role in breaking down peptide bonds within proteins. In the food industry, microbial proteases find diverse applications, including cheese-making, meat tenderization, gluten modification, and protein hydrolysis. Their preference over animal and plant proteases stems from their stability, specificity, and diversity. This research focuses on the extraction, purification, and activity assessment of the protease enzymes obtained from soil and water samples collected at Khulna University. The selection process involved the use of Skim Milk Agar media to identify protease-producing bacteria, including Lysinibacillus sphaericus MGB1077, Bacillus firmicutes HBB525, Bacillus cereus VKK8, Lysinibacillus fusiformis S1B12, and Bacillus cereus CEN5. Submerged fermentation was employed to produce protease from these strains over 48 hours. After centrifugation, we obtained crude protease, which was tested by Sigma’s non-specific protease activity assay using casein as the substrate. Subsequent purification involved 60% ammonium sulfate precipitation. Notably, partially purified proteases exhibited higher activity than crude proteases. We optimized the enzyme production process for the five isolates, resulting in activities of 32.13 Units/ml, 28.42 Units/ml, 18.83 Units/ml, 24.11 Units/ml, and 18.55 Units/ml, respectively. Sodium DodecylSulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) analysis revealed molecular weights of approximately 45 kDa for enzymes from L. sphaericus MGB1077 and B. firmicutes HBB525, while those from B. cereus VKK8 and B. cereus CEN5 exhibited weights around 30 kDa. Additionally, we characterized the enzymes based on incubation time, pH, and temperature. Finally, these proteases were applied to meat, demonstrating a significant tenderizing effect.