Moving away from crude oil to renewable resources for the production of a wide range of compounds is a challenge for future generations. To overcome this, the use of lignocellulose as substrate can contribute to drastically reduce the consumption of crude oil. In this study, sugars from lignocellulose were used as a starting material for the enzymatic synthesis of surface-active sugar esters. The substrates were obtained by an acid-catalyzed, beechwood pretreatment process, which resulted in a fiber fraction that is subsequently hydrolyzed to obtain the monosaccharides. After purification and drying, this glucose- and xylose-rich fraction was used to create a deep eutectic solvent, which acts both as solvent and substrate for the lipase-catalyzed reaction at the same time. Finally, the successful synthesis of glycolipids from a sustainable resource was confirmed by ESI-Q-ToF mass spectrometry and multidimensional NMR experiments. Moreover, conversion yields of 4.8% were determined by LC-MS/MS.