Human diets are rich in secondary metabolites such as polyphenols. These compounds perform a wide range of crucial functions in biological systems and are of great interest for the pharmaceutical and food industries. In this work, the binding mode of the natural polyphenolic compounds from grape seed on the porcine pancreatic elastase surface was studied by experimental and computational methods. Fluorescence quenching, circular dichroism, nephelometry, dynamic light scattering (DLS), molecular docking, and molecular dynamics simulation studies were performed. A decrease in fluorescence intensities was observed with addition of increasing polyphenol concentrations. The order of binding ability obtained was oligomeric fraction of procyanidins (OFP) > tetramer > trimer > dimer B3 procyanidins. Thus a relationship between higher molecular weight and binding ability was observed. The interaction between these molecules and the enzyme occurs by a static mechanism, as inferred from the high apparent fluorescence and bimolecular quenching constants. A blue shift in the maximal emission wavelength could be seen, which indicates that the tryptophan residues acquire a more hydrophobic character upon procyanidin binding. Molecular docking and dynamics simulations also demonstrate that the SASA (solvent-accessible surface area) values of tryptophans decrease with the binding of these compounds, preventing the accessibility of water molecules, which agrees with the referred blue shift. Circular dichroism studies indicate a decrease in alpha-helix content, followed by an increase in the beta-sheet component of secondary structures of this enzyme. DLS and nephelometry techniques also indicate a relationship between large procyanidins and aggregate formation ability.