A three-year experiment was carried out in Central Greece to assess the use of different tillage practices (Conventional, Reduced, and No tillage) for seedbed preparation, in a double cropping per year rotation of irrigated and rainfed energy crops for biomass production for first- and second-generation biofuel production. A life cycle assessment (LCA) study was performed for the first year of crop rotation to evaluate the environmental impact of using different tillage practices, identifying the processes with greater influence on the overall environmental burden (hotspots) and demonstrating the potential environmental benefits from the land management change. LCA results revealed that fertilizer application and diesel fuel consumption, as well as their production stages, were the hot-spot processes for each treatment. In the present study, different tillage treatments compared using mass- and area-based functional unit (FU), revealing that reduced tillage, using strip tillage for spring crop and disc harrow for winter crops, and no tillage treatment had the best environmental performance, respectively. Comparison between the prevailing in the area monoculture cotton crop with the proposed double energy crop rotation adopting conservation tillage practices, using mass and energy value FU, showed that cotton crop had higher environmental impact.