A holistic approach for the optimum exploitation of biomass resources for food, energy and other products will provide the key for a sustainable bio-based future in EU. The small and medium scale applications are expected to play a crucial role in this direction in the future, due to their flexibility and adaptability to the regional and local conditions. The increasing biomass demand and socio-environmental concerns for bioenergy applications require the development of a framework of “Best Practices” for any new application entering the system, in order to ensure its techno-economic feasibility and socio-environmental sustainability. In the present work such a framework will be developed and presented, and it will be specified for the two-step fermentative biohydrogen generation technology. Under this point of view, a “Best Practice” guide is developed for each of the 4 previously selected, most suitable for the examined technology feedstocks, i.e. sugar beet, potato steam peels, barley straw and wheat bran. An outline with main points referring to both sugar beet and barley straw is presented in this work.