Pathological amyloids associated with Parkinson and Alzheimer diseases have been shown to catalyze chemical reactions in vitro. To elucidate how small-molecule substrates interact with cross-β amyloid structures, we here employ computational approaches to investigate α-synuclein amyloid fibrils of the type-1A fold. Our initial binding pocket prediction analysis identified three distinct substrate-binding sites per protofilament, yielding a total of six sites in the dimeric type-1A amyloid structure. Molecular docking of the model phosphoester substrate para-nitrophenyl phosphate (pNPP), previously shown to be dephosphorylated by α-synuclein amyloids in vitro, was performed on the three identified sites. Docking was validated by molecular dynamics simulations for a period of 100 ns. The results revealed a pronounced preference for a single binding site (termed Site 2), as pNPP migrated to this region when primarily placed at the other two sites. Site 2 is located near the interface between the two protofilaments in a cavity enriched with lysine residues and histidine-50. Binding site analysis suggests stable, yet dynamic, interactions between pNPP and these residues in the α-synuclein amyloid fibril. Our work provides molecular-mechanistic details of the interaction between a small-molecule substrate and one α-synuclein amyloid polymorph. This framework may be extended to other reactive substrates and amyloid polymorphs.