Berberine (BBR) and dihydroberberine (HBBR) were investigated as inhibitors of pancreatic lipase in an attempt to explore their potential hypolipidemic activities. The study included docking simulations and in vitro enzymatic inhibition assays. At the molecular level, docking simulations revealed several significant binding interactions between the docked natural compounds and the key amino acids in the binding pocket of the pancreatic lipase enzyme. BBR had similar pattern of binding interactions as HBBR; however, BBR has a permanent cationic center which is suggested to have an adverse influence on ligand–pancreatic lipase affinity. This trend is explainable by the proposition that ionized ligands favor hydration instead of docking into the binding site. This might explain the lower inhibitory activity of BBR comparing to HBBR, which appeared from their estimated IC50 values. The logarithmic regression of PL inhibition versus concentration revealed estimated IC50 values of 106 and 8.0 lg/mL for BBR and HBBR, respectively.