We present an approach where biomolecular self-assembly is used in combination with lithography to produce patterns of metallic nanoparticles on a silicon substrate. This is achieved through a two-step method, resulting in attachment of nanoparticles on desired sites on the sample surfaces, which allowed a detailed characterization. First, a genetically modified hydrophobin protein, NCysHFBI, was attached by self-assembly on a hydrophobic surface or a surface patterned with hydrophobic and hydrophilic domains. The next step was to label the protein layers with 17.8 nm gold nanoparticles, to allow microscopic characterization of the films. Kinetics and extent of attachment of nanoparticles were characterized by UV−vis spectroscopy and transmission electron microscopy. It was shown that the attachment of citrate-stabilized gold nanoparticles was strongly dependent on the electrostatic properties of the capping ligand layer and the density of nanoparticles in the monolayer could be controlled via pH. The resulting nanoparticle assemblies followed the original pattern created by optical lithography in high accuracy. We demonstrate that combining bottom-up and top-down nanotechnological approaches in a good balance can provide very effective ways to produce nanoscale components providing a functional interface between electronics and the biological world.