The self-assembly of heterogeneous bilayers on Au substrates was investigated using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and infrared reflection absorption spectroscopy (IRAS). The formation of a well-defined bilayer including different types of functional groups would be one of the desired goals to create varying surface functionalities. In this study, we examined the assembly of a hydrogen-bonded molecular layer to another functional alkanethiolate self-assembled monolayer (SAM) on the Au(111) surface. The chemical properties and bond strength of the hydrogen bonds at the interlayer differ from those of the Au–S bonds at the anchor of thiolate SAMs, therefore the adsorbed molecules are expected to form a stratified bilayer. In this study, on one hand, we revealed that imidazole-terminated alkanethiolate SAMs (Im-SAMs) have an atomically smooth topography but chemically inhomogeneous Au–S anchors, rather incomplete than n-alkanethiolate SAMs, on the Au(111) surface. On the other hand, we confirmed the self-assembly of the heterogeneous bilayers including Im-SAMs on the Au(111) surface, even in a mixed solution containing two types of molecules. These results show that the self-assembly of the bilayer stratified by H bonds and Au–S bonds is flexible and adaptable.