Background and Objectives: The use of ascorbic acid together with copper ions as disinfectant is based on the findings that during oxidation of ascorbic acid in the presence of copper ions, some compounds with antibacterial properties are produced which are able to inactivate some bacterial spores. Further investigation showed that the presence of dissolved oxygen is essential in this process. The aim of this study is to evaluate performance of copper - ascorbic acid and dissolved oxygen combination in disinfection of secondary treated wastewater. Methods: this study performed with injection of different doses of disinfectants in presence and absence of pressurized oxygen. Iranian environment protection agency and world health organization (WHO) standards and guidelines for concentration of Thermotolerant Coliforms in discharged effluents are used as disinfection target level. Results: the results of this study showed that using copper ions in combination with ascorbic acid in conventional doses meets the determined disinfection target level. Applying pressurized dissolved oxygen fundamentally increased the system. 3Cu + 50AA mg/L in both presence and absence of pressurized dissolved oxygen met the WHO guideline however, Iranian EPA standard was met only in last dose (5Cu + 100AA) in presence of O2. Conclusion: This study was done in pilot scale. Application of copper-ascorbic acid in presence of pressurized dissolved oxygen can inactive Termotelorant Coliforms of natural samples of secondary wastewater to acceptable level.