Bacterial biofilms are a persistent source of contamination, and much effort has been invested in developing antifouling surfaces or coatings. A bottleneck in developing such coatings is often the time-consuming task of screening and evaluating a large number of surface materials. An automated high-throughput assay is therefore needed. In this study, we present a promising technique, laser scanning cytometry (LSC), for automated quantification of bacteria on surfaces. The method was evaluated by quantifying young Staphylococcus xylosus biofilms on glass surfaces using LSC and comparing the results with cell counts obtained by fluorescence microscopy. As an example of application, we quantified bacterial adhesion to seven different sol-gel-based coatings on stainless steel. The surface structure and hydrophobicity of the coatings were analyzed using atomic force microscopy and water contact angle measurements. Among the coatings tested, a significant reduction in adhesion of S. xylosus was observed only for one coating, which also had a unique surface microstructure. LSC was particularly sensitive for quantification at low cell densities, and the adhered bacteria could be quantified both as cell number and as area coverage. The method proved to be an excellent alternative to microscopy for fast and reproducible quantification of microbial colonization on abiotic surfaces.