A small library of 22 N-substituted galactonoamidines was synthesized, and their structure-activity relationship for inhibition of the hydrolytic activity of β-galactosidase (Aspergillus oryzae) was evaluated. A fast screening assay in 96-well plate format was used to follow the enzymatic hydrolysis of 2-chloro-4-nitrophenyl-β-D-galactopyranoside using UV-vis spectroscopy. The aglycon moiety of all compounds was found to have a profound effect on their inhibitory ability. In general, galactonoamidines derived from cyclic aliphatic and linear amines show higher inhibition activity than those derived from benzylamines. Hydrophobic interactions of the methyl group rather than π-π stacking interactions of the aromatic ring in p-methylbenzyl-D-galactonoamidine were identified to cause its transition-state-like character and the remarkably high inhibitory ability (K(i) = 8 nM). A flexible 3-carbon methylene spacer between the exo N atom of the sugar moiety and a phenyl group furthermore increased the observed apparent inhibition drastically.