Xylo-oligosaccharides, produced from lignocellulosic materials (LCB), are short-chain polymers with prebiotic activity who, in the last decades, have gained commercial interest due to its potential applications as an ingredient to the nutraceutical industry. In this work xylan extraction at different conditions was evaluated from sawdust from four different Chilean native trees. Among them, the highest xylan recovery yields (over 60%) was obtained employing Nothofagus pumilio, selecting this hardwood as the best raw material. On the other hand, to select the best enzyme for XOS production, a commercial and a novel recombinant xylanase were characterized in terms of endoxyanase activity and thermal stability. The selected biocatalyst was employed in the hydrolysis of pre-extracted xylan from Nothofagus pumilio for the enzymatic production of XOS.