There has been an ongoing debate about whether water enhances or hinders π−π stacking, a phenomenon crucial in various biological and chemical systems. In this study, the influence of water on π−π stacking is investigated by microwave spectroscopic observation of gas-phase hydrated clusters of thiophene dimers. Two isomers of (C 4 H 4 S) 2 -H 2 O and two isomers of (C 4 H 4 S) 2 -(H 2 O) 2 have been unambiguously identified. These identifications are supported by quantum chemistry calculations and isotopic measurements. In each of these conformations, water molecules are situated between aromatic pairs, forming distinctive interactions. Water molecules engage with thiophene molecules either as hydrogen bond donors through OH•••π interactions or as hydrogen bond acceptors through CH•••O interactions. The energy decomposition analysis indicates that the bonding pattern of water molecules significantly affects the π•••π interactions between aromatic rings. These findings offer valuable structural insights into the role of water in shaping π−π stacking.