The specific structure of dental units favours the presence of biofilm and microbial contamination of the dental unit waterlines (DUWL) water. The ability of bacteria to colonize surfaces and to form biofilm in water supply tubes, including DUWL, is a common phenomenon, which has been well documented, just as with difficulties in biofilm removal and prevention of its regrowth. Microorganisms from contaminated DUWL are transmitted with aerosol and splatter, generated by working unit handpieces. On the basis of the detailed literature review, the state-of-the art knowledge of the microflora of dental unit waterlines is presented. Most of the microorganisms isolated from DUWL are of low pathogenicity. Nevertheless, the public health significance of many of the microorganisms found in DUWL is unknown. According to current knowledge, it is not the mere presence of bacteria that is important in DUWL contamination monitoring, but their number, the presence of potential pathogens, and patients' oral cavity microflora. Numerous studies emphasize the need for effective mechanisms to reduce the microbial contamination in DUWL and highlight the risk for cross-infection in general practice, especially in view of the ever-increasing number of immunocompromised persons who present at outpatient dental clinics.