Waterborne Polyurethanes (PUs) are a family of polymers that contains urethane linkages synthesized in an aqueous environment and are thus free of organic solvents. Recently, waterborne PUs have been extensively studied for biomedical applications because of their biocompatibility. The present work investigates the following: (1) the impact on electrical performance of electrode materials (platinum and silicon) modified chemically by a layer of waterborne PU, and (2) the behavior of rat cardiac fibroblasts and rat cardiomyocytes when in contact with an electrode surface. Diisocyanate and poly(caprolactone diol) were the main reagents for producing PUs. The electrochemical impedance of the electrode/electrolyte interface was accessed by electrochemical impedance spectroscopy. The cellular viability, proliferation, and morphology changes were investigated using an MTT assay. Cardiomyocyte adherence was observed by scanning electron microscopy. The obtained surface was uniform, flat, and transparent. The film showed good adhesion, and no peeling was detected. The electrochemical impedance decreased over time and was influenced by the ionic permeability of the PU layer. The five samples did not show cytotoxicity when in contact with neonatal rat cells.