Purified lipase fractions from crude commercial extract of Aspergillus niger lipase (ANL) were selectively immobilized on hydrophobic supports. A significant percentage of esterase activity remains in the supernatant, derived from esterase(s) unable to become adsorbed onto the employed matrices. These residual proteins were covalently immobilized on epoxy-acrylic resin. Immobilized hydrolases were tested in the hydrolysis of acetylated disaccharides in water-cosolvent systems. ANL-Esterase was able to catalyze regioselective deprotection of acetylated β-O-methyl lactoside in C-2 position and β-O-methyl lactosaminide in C-3′ position. The hydrolyzed products, never reported before, can be considered new building blocks for the synthesis of oligosaccharides of biological relevance. Furthermore, preparative hydrolyses were also performed in tert-butanol. This solvent is compatible with ANL-esterase stability and it appears to be a novel and promising approach because of its green status.