Abstract An evaluation of the investment required to implement a second generation bioethanol production chain is presented in this study. An integrated agro-industrial system can be an appropriate solution to accomplish the European requirement for the partial substitution of bioethanol for gasoline by 2020. A biorefinery system is hypothesized within the Province of Siena (Italy), fed by residual energy and material flows from local productions: straw from agriculture and residual geothermal heat from geothermal electricity generation; the output is calibrated to replace 10% of gasoline consumption within the Provincial area. The physical consistency of the investment required to implement the production process, as well as the benefit of the biofuel–gasoline substitution, have been evaluated by means of the emergy methodology. Emergy is a thermodynamics-based indicator that contributes to identify and measure all the inputs supporting a given system, expressed in a common unit (solar emergy Joules – semj). Results show that the benefit of saving gasoline, in emergy terms, almost doubles the emergy investment to produce biofuel. The case of a biorefinery fed by natural gas instead of local residual geothermal heat is also presented. The Unit Emergy Investment (UEI) of bioethanol produced in a biorefinery that uses residual straw and geothermal heat is 9.16E + 08 semj/g (the case of the Province of Siena); it is 1.84E + 09 semj/g (in case natural gas is used to fuel the process). Finally, the comparison between the UEI of bioethanol evaluated in this study and UEV of bioethanol calculated in other studies is presented.