Spatial control of surface functionalization and interactions is essential for microarray-based analysis. This study reports the fabrication of two-dimensional molecular films with site-specific functionalities, forming microarrays at discrete locations. Arrays of microsized gold disks were produced on a silicate membrane using microfabrication. On these arrays, orthogonal self-assembly of molecules was performed that can specifically bind to gold or silicate. The gold array elements were functionalized with a range of alkanethiols and the silicate with polymeric poly-L-lysine-grafted-poly(ethylene glycol) (PLL-PEG). The surface functionalization on the gold disk array and the surrounding substrate was characterized at each step using X-ray photoelectron spectroscopy (XPS) to show that alkanethiols are specifically attached to the gold. PLL-PEG was used to provide resistance against nonspecific protein and cell adsorption and attached exclusively to the silicate. The effectiveness of the surface chemistry was validated by the selective self-assembly of a gold nanoparticle monolayer array on the gold regions. In a more sophisticated example, selective adhesion of MCF-7 cells to anti-EpCAM antibody modified gold areas of the gold-silicate surface was demonstrated to give a cell microarray. This study provides a general approach to fabricate chemical patterns on silicon-based devices with the convergence of microfabrication and material-specific surface modification, which may be useful to expand the functionalities and potential applications for patterned biomolecular films. Importantly, the ability to pattern surfaces with different surface chemistries is not limited to planar surfaces using this orthogonal surface-coupling approach.