Floating lipid bilayers composed of phosphatidylglycerols and cardiolipin were deposited on gold electrodes premodified with 1-thio-β-d-glucose monolayer by spreading of small unilamellar vesicles. The resulting lipid membrane was homogeneous, and its thickness was ∼5.0 nm. Electrochemical characterization combined with surface-enhanced infrared absorption spectroscopy revealed that negative polarization of the electrode leads to accumulation of water molecules in the interfacial region between lipid membrane and the thioglucose film. Moreover, the buildup of water layer was demonstrated to affect the nanomechanical properties of the membrane. The latter was manifested by well-pronounced decrease of Young's modulus of the lipid bilayer correlating with increasing hydration. This effect was ascribed to the decoupling of the membrane from supporting thioglucose film due to the accumulation of interfacial water. As a result, the effective stiffness of the supporting layer is lower and it alters the nanomechanical behavior of lipid membrane. Our results provide strong experimental proof for the correlation between elastic properties of floating lipid membrane and the amount of water accumulated in the submembrane region.