Abstract: Background and Objective: Bacillus subtilis is a bacterium that can produce the oxalate decarboxylase (ODC) enzyme. This enzyme decomposes oxalate. ODC enzyme activity is influenced by temperature and pH. This study was conducted to determine the optimum pH and temperature for ODC enzyme activity in B. subtilis FNCC 0059. Materials and Methods: B. subtilis was cultivated in oxalate medium at different incubation temperatures for 24 h. The tested temperatures were 25, 29, 33, 37, 41 and 45°C. In separate experiments, cultures were grown in oxalate medium at varying pH from 4-8.5. The cell growth assay and enzyme activity were performed and all the experiments were done with 3 replicates. Data were analyzed using one-way analysis of variance. Results: The optimum temperature of 37°C could produce maximum ODC enzyme activity (0.041 U mL–1) enzyme activity increased to the point of the optimum pH and then decreased 5.6% when the pH was increased 0.5 from the optimum. Maximum ODC enzyme activity (0.0413 U mL–1) was achieved at pH 5.5. Conclusion: Temperature and the pH of the medium affected ODC enzyme activity. Optimum medium condition of B. subtilis was reached at 37°C and pH 5.5.