Protease-producing bacteria were isolated from freshwater samples collected previously from the Rosetta branch (Nile River, Egypt) and were identified as Bacillus subtilis and Pseudomonas aeruginosa. The optimization of the culture conditions as pH, incubation time, substrates, carbon, and nitrogen sources were applied for maximizing the production of protease enzyme as well as the application of enzyme partial purification and enzyme characterization to study the effects of pH, temperatures, and different types of metals. The results referred to the best conditions for protease production of Bacillus subtilis were found to be gelatin substrate, pH 7.5, within 30 hr. incubation period, and the better source of carbon and nitrogen was lactose and peptone respectively. On the other side, the best conditions for Pseudomonas aeruginosa were found to be casein substrate, pH 8, within 48 hr. incubation period, and the better source of carbon and nitrogen was galactose and peptone respectively. The maximal activities at pH 9 and 8 for partially purified enzymes extracted from Bacillus subtilis and Pseudomonas aeruginosa respectively within incubation temperature at 40°C. Finally, Co²⁺ is the best metal ion that enhanced proteolytic activity for enzymes extracted from Bacillus subtilis, while the activity of the enzyme extracted from Pseudomonas aeruginosa was enhanced by Zn²⁺, Ca²⁺, and Co²⁺. Among all studies for bacterial isolates, the highest activity of protease enzyme was observed in Bacillus subtilis.