Aquatic biofilms, which are well-organized communities of microorganisms, are widespread in nature. They constitute a major problem in many environmental, industrial and medical settings. The use of advanced techniques has revealed biofilm structure, formation and ecology. Special attention was given to the build-up of biofilm in dental unit waterlines (DUWLs), which are small-bore flexible plastic tubing to bring water to different handpieces. They are coated with well-established biofilms. Active biofilm is a source of microbial contamination of DUWLs water. The safety of dental treatment requires a good quality of the water used. The knowledge of nature, formation and the ways to eliminate the biofilm is the first step towards reducing health risk, both for patients and dental personnel. The article reviews these issues.