This work investigates the molecular interactions between Agaricus bisporus lectin (ABL) and the dietary carbohydrates, glucose and galactose, in comparison to their N-acetylated amino sugar counterparts. Intrinsic fluorescence quenching suggests the presence of binding between ABL and each of the investigated ligands (glucose, galactose, N-acetyl-d-glucosamine and N-acetyl-d-galactosamine), with the former exhibiting the strongest interaction. Molecular docking highlights the likely binding positions, showing the presence of stable interactions between protein and ligands. The conformational difference of a single epimeric hydroxyl group at carbon four of the sugar ring appears to be the main factor in determining binding location, with galactose containing molecules favouring the T-antigen binding site whereas glucose containing molecules favouring another binding location. Fourier transform infrared (FTIR) and circular dichroism (CD) record spectral changes, suggesting that the heterologous interactions affect the secondary structure of the protein molecule. The magnitude of structural alteration in the complex is related to the binding strength. Findings provide a theoretical basis for the potential application of ABL in functional foods and hypoglycaemic nutraceuticals, as well as contributing to the fundamental understanding of the molecular mechanism behind ABL and dietary carbohydrate complexation.