Interactions between fulvic acids (FAs) and a β-glucosidase (GLU) enzyme and consequent modifications of enzymatic activity were investigated at pH 5.0 and 7.2 by 1H nuclear magnetic resonance (NMR) spectroscopy. With increasing FA content, the enzyme proton signals were progressively broadened, while the relaxation (T1 and T2) and correlation (τc) times of GLU decreased and increased, respectively. Regardless of pH, these effects were greater for the hydroxy-alkylic and aromatic protons of GLU, suggesting that the FA-enzyme associations, which progressively limited GLU tumbling rate, resulted from weak interactions, such as H-bonds and dispersive hydrophobic bonds. The catalytic activity of β-D-glucosidase, when in weakly bound complexes with FA, was studied by following the change of NMR signals of two different substrates, p-nitrophenyl-β-D-glucopyranoside (pNPG) and salicin, and their hydrolysis products. Spectral evidence suggests that enzyme activity was substantially reduced with increasing FA concentration and the rate reduction was more pronounced for salicin than for pNPG. The enzyme inhibition may be explained by either a partial cover of GLU active sites by fulvic molecules or modification of the enzyme conformational structure during formation of humic-enzyme complexes. Our results indicate that even weak interactions of FA with GLU are sufficient to inhibit partially its catalytic activity, and that the environmental role of extracellular enzymes may be significantly reduced when coming into contact with organic matter in the soil.