A glycerol-organosolv process can be a good alternative for Eucalyptus wood fractionation into its main compounds, improving the enzymatic saccharification of the cellulose. A study of process variables - glycerol-water percent content, temperature, and process time - was carried out using a Box-Behnken experimental design. The cellulose obtained from pretreated solids was recovered almost quantitatively, leading to a solid with a high percentage of cellulose (77 g/100 g of pretreated solid), low lignin content (9 g/100 g of pretreated solid), and 18% of residual hemicellulose in the solid at 200 degrees C, 56% of glycerol-ater and 69 min. The enzymatic saccharification was enhanced achieving 98% cellulose-to-glucose conversion (under conditions: liquid to solid ratio 20 g/g and enzyme loading 20 FPU/g of solid). This study contributes to the improvement of biomass fractionation by exploring an eco-friendly treatment which allows for almost complete wood fractionation into constituents an high levels of glucose recovery available for subsequent yeast fermentation to bioethanol.