Xylanases play a crucial role in the hydrolysis of xylan-rich hemicelluloses and have wide industrial applications in the fuel, food, feed and pulp and paper industries. The production of these enzymes at low cost is of paramount importance for their commercial deployment. Moesziomyces antarcticus PYCC 5048T and M. aphidis PYCC 5535T were screened for their ability to produce xylanolytic enzymes when grown on d-xylose, xylan (beechwood) and brewery's spent grain (BSG). The extracellular crude extracts produced were characterized and tested in xylan hydrolysis. The yeasts produced xylanolytic enzymes without cellulolytic activity on all the substrates tested. The highest xylanase volumetric activity was obtained with M. aphidis PYCC 5535T grown on BSG, reaching 518.2 U/ml, a value 8.4- and 4.7-fold higher than those achieved on xylan and d-xylose, respectively. The xylanase activities were characterized in relation to pH and temperature with optima at 4.5 and 50 °C, respectively. The extracts from both M. antarcticus PYCC 5048Tand M. aphidis PYCC 5535T were used in xylan hydrolysis, producing d-xylose as the major end product (0.43 and 0.34-0.47 gD-xylose/gxylan, respectively, at 50 °C) and relatively low or no xylobiose accumulation (from no detection to 0.12 gD-xylobiose/gxylan at 50 °C).