In Thailand, the Alternative Energy Development Plan has set the target to increase the use of bioethanol to 9.00 million liters per day by 2021. To achieve this goal, both freshwater availability for energy crops and best practices in bioethanol production chain management are very important issues. Therefore, this study integrates water footprint technique with the linear programing approach in order to optimize the operations decision, focusing on water footprint of the bioethanol production chains from both tactical and operational levels. A cradle-to-grave approach is adopted to evaluate the water consumption and pollution in bioethanol production from sugarcane and cassava. The results show that the water footprint of bioethanol consumed in Thailand was about 3.23 × 109, 1.72 × 1010, and 2.49 × 1010 m3 per year in 2010, 2016, and 2021, respectively. The share of agriculture water consumption to the total water footprints of bioethanol was 99% and industrial water consumption was 1%. After applying the linear programing, it was found that the water footprint could be reduced by at least 53%, or 1.33 × 1010 m3, annually. The modeling approach and formulation presented could be used as a tool to reduce water consumption and provide the operation plan of bioethanol production chain.