Microbial proteases, widely used in various industrial processes and commercial applications, are hydrolytic enzymes of significant importance. This study aimed to isolate and characterize efficient protease-producing bacteria from soil samples collected in Mardan. Screening for proteolytic activity on skim milk agar and casein agar yielded six potent bacterial isolates. These were identified as five Bacillus species and one cocci species based on morphological and biochemical characteristics. The optimum protease production time for the isolates was determined to be 24 hours, with proteolytic activities ranging from 4 mm to 12 mm. The isolate P6 exhibited the highest activity (12 mm), while P11 showed the least (4 mm). Optimal conditions for protease activity were found to be 37°C and pH 9 for all isolates, with activity declining at higher temperatures. Substrate specificity tests using casein, soybean flour, and gelatin revealed that the isolates demonstrated superior proteolytic activity on soybean flour and casein. The effect of various additives on protease production was also investigated. Media supplemented with MgCl2 resulted in maximum proteolytic activity compared to other metallic ions tested (CaCl2 and NaCl). However, NaCl was found to be effective for some isolates, particularly P6 Bacillus sp., which consistently showed the highest protease activity across all parameters tested. This study identifies several promising protease-producing bacterial strains, with P6 Bacillus sp. emerging as the most potent. Further characterization and purification of these enzymes at the molecular level are recommended for their potential industrial applications.