Biofilms are an important source of contamination in food companies, yet the composition of biofilms in practice is stillmostly unknown. The chemical and microbiological characterization of surface samples taken after cleaning and disinfection isvery important to distinguish free-living bacteria from the attached bacteria in biofilms. In this study, sampling methods that arepotentially useful for both chemical and microbiological analyses of surface samples were evaluated. In the manufacturingfacilities of eight Belgian food companies, surfaces were sampled after cleaning and disinfection using two sampling methods:the scraper–flocked swab method and the sponge stick method. Microbiological and chemical analyses were performed on thesesamples to evaluate the suitability of the sampling methods for the quantification of extracellular polymeric substancecomponents and microorganisms originating from biofilms in these facilities. The scraper–flocked swab method was mostsuitable for chemical analyses of the samples because the material in these swabs did not interfere with determination of thechemical components. For microbiological enumerations, the sponge stick method was slightly but not significantly moreeffective than the scraper–flocked swab method. In all but one of the facilities, at least 20% of the sampled surfaces had more than102 CFU/100 cm2. Proteins were found in 20% of the chemically analyzed surface samples, and carbohydrates and uronic acidswere found in 15 and 8% of the samples, respectively. When chemical and microbiological results were combined, 17% of thesampled surfaces were contaminated with both microorganisms and at least one of the analyzed chemical components; thus, thesesurfaces were characterized as carrying biofilm. Overall, microbiological contamination in the food industry is highly variable byfood sector and even within a facility at various sampling points and sampling times.