Control of molecular self-assembly at solid− liquid interfaces is challenging due to the complex interplay between molecule−molecule, molecule−surface, molecule− solvent, surface−solvent, and solvent−solvent interactions. Here, we use in-situ dynamic atomic force microscopy to study the self-assembly of Benzopurpurine 4B into oblong islands with a highly ordered inner structure yet incommensurate with the underlying calcite (10.4) surface. Molecular dynamics and free energy calculations provide insights by showing that Benzopurpurine 4B molecules do not anchor to the surface directly but instead assemble on top of the second hydration layer. This seemingly peculiar behavior was then rationalized by considering that hydrophobic molecules placed atop the second water layer cause the least distortion to the existing hydration structure. Further experiments for the adsorption of Benzopurpurine 4B on other minerals indicate that the specific interfacial water structure on calcite is decisive for rationalizing the self-assembly of Benzopurpurine 4B in this system.