Abstract A cradle-to-wheel life cycle analysis evaluated the greenhouse gas (GHG) contribution and fossil energy consumption (FEC) of bio-ethanol (EtOH) from dedicated crops on marginal lands in Campania Region (Southern Italy). The analysis processed experimental agronomic data of giant reed cultivated on hilly areas at high erosion risk and industrial data of feedstock conversion to EtOH, through second generation innovative technologies. Two ethanol–gasoline mixes (E10 and E85) were considered and compared with gasoline vehicle (GV). The E10 GHG and FEC profiles were driven by the gasoline input in the blend and the linked CO2 tailpipe emissions. The EtOH supply chain, especially the crop phase, was the major contributor to E85 impacts. The higher amount of biogenic C in E85 determined a marked reduction over GV of both GHG emissions (−60%) and FEC (−65%). The soil carbon storage would save 25 g CO2 eq per MJ; however, the issue is controversial due to the spatial and temporal variability of the process. Based on land availability, E85 contribution to renewables in the regional transport sector would amount to 4% and the blend wall would be far from saturation. A possible mismatch between future flexi-fuel vehicles density and E85 availability should be considered.