The contribution of agrobiomasses, as a source of energy, to the reduction of greenhouse gas emissions was confirmed by several studies. Biomass from agriculture represents one of the larger and more diverse sources to exploit and in particular ethanol and diesel have the potential to be a sustainable replacement for fossil fuels, mainly for transport purposes. However, the cultivation of energy crops dedicated to the production of biofuels presents some potential problems, e.g., competitiveness with food crops, water needs, use of fertilizers, etc., and the economic, energy, and environmental convenience of such activity depends on accurate evaluations about the global efficiency of the production system. In this study, the processes related to the cultivation of energy crops were analyzed from an energy and water cost perspective. The crops studied, maize (Zea mais) and sunflower (Helianthus annuus), were identified for their different water requirements and cultivation management, which in turns induces different energy costs. A 50-year climatic series of meteorological data from 19 weather stations scattered in the Tuscany region was used to feed the crop model CropSyst for the simulation of crop production, water requirement, and cultivation techniques. Obtained results were analyzed to define the real costs of energy crop cultivation, depending on energy and water balances. In the energy crop cultivation, the only positive energy balance was obtained with the more efficient system of irrigation whereas all the other cases provided negative balances. Concerning water, the results demonstrated that more than 1.000 liters of water are required for producing 1 liter of bioethanol. As a consequence, the cultivation of energy crops in the reserved areas of the region will almost double the actual water requirement of the agricultural sector in Tuscany.