Background: Water quality in the drinking water system (DWS) plays an important role in the general health andperformance of broiler chickens. Conditions in the DWS of broilers are ideal for microbial biofilm formation. Sincepathogens might reside within these biofilms, they serve as potential source of waterborne transmission ofpathogens to livestock and humans. Knowledge about the presence, importance and composition of biofilms inthe DWS of broilers is largely missing. In this study, we therefore aim to monitor the occurrence, and chemicallyand microbiologically characterise biofilms in the DWS of five broiler farms.Results: The bacterial load after disinfection in DWSs was assessed by sampling with a flocked swab followed byenumerations of total aerobic flora (TAC) and Pseudomonas spp. The dominant flora was identified and theirbiofilm-forming capacity was evaluated. Also, proteins, carbohydrates and uronic acids were quantified to analysethe presence of extracellular polymeric substances of biofilms. Despite disinfection of the water and the DWS, averageTAC was 6.03 ± 1.53 log CFU/20cm2. Enumerations for Pseudomonas spp. were on average 0.88 log CFU/20cm2 lower.The most identified dominant species from TAC were Stenotrophomonas maltophilia, Pseudomonas geniculata andPseudomonas aeruginosa. However at species level, most of the identified microorganisms were farm specific. Almostall the isolates belonging to the three most abundant species were strong biofilm producers. Overall, 92% of all testedmicroorganisms were able to form biofilm under lab conditions. Furthermore, 63% of the DWS surfaces appeared tobe contaminated with microorganisms combined with at least one of the analysed chemical components, which isindicative for the presence of biofilm.Conclusions: Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa are considered asopportunistic pathogens and could consequently be a potential risk for animal health. Additionally, the biofilm-formingcapacity of these organisms could promote attachment of other pathogens such as Campylobacter spp. andSalmonella spp.