Since the operating conditions of the cooling tower units provide such a suitable environment for the growth of bacteria and algae, taking measures for the disinfection process is strongly recommended. The cooling tower unit is proceeded by RO and the disinfection strategy is sodium hypochlorite, combined with this new formulation. The sampling result revealed that bacteria counts from the effluent of the cooling tower are 2600 CFU/ml while the microbial load of the water in the pond is 220 CFU/ml. Herein, two type formulations are introduced for cooling tower output stream while the pond water comes along with a NaOCl injection. The dosage of A: Glucose Oxidase, B: ammonium persulfate, and C: Amoxicillin, and D: the order of use are chosen as the four independent variables whose effects and their binary interactions on microbial load and disinfection efficiency were investigated via Box–Behnken design (BBD) combined with response surface methodology (RSM). The ANOVA results show that the most significant variable is amoxicillin in the presence of bleach. Meanwhile, high values for R2 > 0.99 and the model F-value 45.64 in addition to diagnostic tests confirmed the reliability of the model. Several solutions have been introduced as optimum formulations. The pre-scale up and economic considerations have been included.