A simple, efficient and mild strategy for the complete O-deacetylation of different per-acetylated biomolecules in aqueous media has been described. Different lipases were tested but only the commercial Amano lipase A from Aspergillus niger catalyzed the complete deprotection of peracetylated α-glucose to glucose in excellent yield. The experimental conditions were tested, in particular the pH effect. The reaction was performed at different pHs considering the only enzymatic process was evaluated at pH 5 and the combination of enzymatic and chemical migration process was evaluated at higher pHs. Finally pH 7 and 25 °C were selected as best conditions. Thus this lipase fully hydrolyzed different peracetylated α-glycopyranosides (glucose, mannose, glucal, galactal) with >99% yields, whereas very good deprotecting yields (75–80%) were achieved for different acetylated β-glycopyranosides (galactose, ribofuranose) under these mild conditions. This strategy was successfully extended to the fully O-selective deprotection of acetylated nucleosides where >99% yield was rapidly obtained. No selectivity was observed for the N-deacetylation in amino acids and peptides.