Biofuel production from agricultural residues (i.e. lignocellulose) has been recently investigated as a renewable energy alternative to current fossil fuels. Lignocellulosic biomass is one of the most important carbon sources, with remarkable potential as raw material for the production of several valuable products (e.g. chemicals, materials and biofuels). The separation of lignocellulose into its major components (cellulose, hemicelluloses and lignin) is a key step in lignocellulosic biorefineries. Most pretreatments of lignocellulosic biomass into chemicals or biofuels are currently based on expensive chemical and energy consuming processes, which entail significant resource consumption (e.g. water) and generate a number of residual streams. In this work, an innovative dry fractionation technologies (physical fractionation: electrostatic separation of lignocellulose particles) have been developed for straw fractionation and bioconversion to sugars and biofuels. Electrostatic fractionation technology (EF-T) is based on the separation of particles according to their surface properties (chemical composition and charges). EF-T is suitable for producing lignocellulose fractions displaying very different structures, biochemical compositions and reactive surfaces without extensively damaging the raw fibers as well as minimizing waste generation (Environmental-factor: 0.7–0.75). The produced fractions could be hydrolyzed, being able to produce large quantities of glucose (250–280 g kg-1) after 72 h of hydrolysis and subsequently ethanol (130–150 g kg-1) after fermentation. EF-T can therefore improve the economic feasibility by low energy consumption and produce reactive lignocellulose particles with different physicochemical structures in a short time, which can be easily converted to biofuels, minimizing waste (no effluent generation).