Nanocomposites from a hexamethylene diisocyanate (HDI)-based polyester-type waterborne polyurethane (PU) containing different amounts (17.4-174 ppm) of gold (Au) nanoparticles (similar to 5 nm) were prepared. The microstructure and physiochemical properties of the nanocomposites were characterized. The cell attachment and proliferation, platelet activation, and bacterial adhesion on the nanocomposites were evaluated. Gold nanoparticles in small amounts induced significant changes in surface morphology and domain structures, from hard segment lamellae to soft segment micelles. These changes resembled the morphological transformation among different mesophases occurred in diblock copolymers. Better cellular proliferation, lower platelet activation, and reduced bacterial adhesion were demonstrated for the PU nanocomposite with 43.5 or 65 ppm of Au than the pure PU or the nanocomposite containing a different amount of Au. The different cellular response on PU-Au nanocomposites was attributed to the extensively modified surface morphology and phase separation in the presence of a small amount of Au nanoparticles.