In the last decades, the use of enzymes for the conversion of lignocellulosic biomass into energy, materials, platform chemicals or bioactive molecules, namely the biorefinery, has emerged as a green alternative for a sustainable economy. Hemicelluloses, the second most abundant source of renewable carbon in plants, is mainly constituted of pentoses and represent an underutilized raw material with a high potential for the production of value-added compounds. Due to the numerous advantages of enzymes when compared to chemical catalysts, the use of hydrolytically-active hemicellulases as synthetic tools in glycosynthesis by reverse hydrolysis or transglycosylation reactions with activated substrates has become a very attractive area of research. This chapter proposes a global overview of the recent developments in the use of pentose-acting glycoside hydrolases: α-l-arabinofuranosidases, β-d-xylosidases and xylanases for the production of various pentosides with potential applications in different fields. A final part is also dedicated to lipases and their uses in the synthesis of pentose-based esters.