Two pyridoacridine alkaloids, including a known petrosamine and a new 2-bromoamphimedine were isolated from a Thai marine sponge Petrosia n. sp. The alkaloids were characterized on the basis of 1D and 2D NMR, MS, and IR spectroscopy. Only petrosamine showed strong acetylcholinesterase inhibitory activity approximately six times higher than that of the reference galanthamine. A computational docking study of petrosamine with the enzyme from the electric eel Torpedo californica (TcAChE) showed the major contribution to the petrosamine-TcAChE interaction to be arising from the quaternary ammonium group of petrosamine.