AbstractSeveral timbers and crop by-products were subjected to an enzymatic treatment to obtain a xylo-oligosaccharides-enriched preparation. The process was performed by means of the commercial endo-β-1,4-xylanase Buzyme 2511®. The enzymatic cocktail was applied onto the raw ground materials with yield up to 5.3 ± 1.0 g/L xylo-oligosaccharides for apple pomace. In order to make the materials more accessible to enzymatic hydrolysis, they were subjected to thermal-alkaline treatment. The biocatalysis process over the thermo-alkaline treated materials yielded xylo-oligosaccharide solutions with the following concentrations (g/l): 1.3 white poplar (Populus alba), 2.9 giant cane (Arundo donax), 3.7 apple pomace (Malus domestica), and 6.5 stalk of grapes (Vitis vinifera). The preparation resulting from biotransformation of grape stalk contained mostly xylo-oligosaccharides (96% w/v) with a small amount of xylose (3% w/v). The same ratio was obtained when pure xylan from birchwood was used as feedstock.