Abstract The Directive 2009/28/EC established the overall target that 20% of energy consumption should be represented by renewable energy sources by 2020 in each European member state. Furthermore, the Directive sets a mandatory 10% minimum target for biofuels in the transport sector. Biofuels are potentially an important alternative to mineral diesel. We propose a pilot production line of biodiesel from sunflower on local scale in the Province of Siena (Tuscany) to research a possible reduction of fossil fuel consumption in the transport sector. This study might represent an opportunity to reduce petroleum dependence in the transport sector. Environmental Impact Indicators were provided by Material Flow Accounting, Embodied Energy Analysis and Emergy Accounting. Results showed that agricultural phase is the critical step in the production line. A comparative Life Cycle Assessment analysis for the biodiesel production line with mineral diesel production showed environmental advantages of the biofuel production, however requiring a higher land demand. Therefore, biodiesel may not the optimal solution on large scale but might be a good alternative to fossil fuel. This would depend upon the entire production cycle taking place in a limited area. This is necessary in order to fulfill the needs of local farms and small enterprises.