Biofilms are communities of microorganisms embedded in a self-produced extracellular matrix made up of polymeric substances. They reduce the effects of antibiotics and allow the microorganisms to evade the innate immune system. This can lead to persistent or recurrent infections. In dairy cow herds, mastitis is a serious problem. The present study aimed to investigate the occurrence of biofilms in the udders of dairy cows infected with Staphylococcus (S.) aureus, because biofilms may affect the response to treatment of bovine mastitis. Immunofluorescence staining of polysaccharide intercellular adhesin (PIA), a component of S. aureus biofilms, was carried out based on swabs taken from different areas of S. aureus infected udders. We were able to demonstrate the presence of PIA in S. aureus infected bovine udders. However, the applied method is invasive and therefore only really suitable for scientific research and not for clinical diagnosis.