Dental Unit Water Systems (DUWS) can be a source of contamination to dentists and patients, especially those who are immunocompromised. Such organisms may originate from incoming water supplies and from oral cavities. The most common cause of dental unit water contamination is believed to be the formation and subsequent sloughing off of microbial biofilms from tubing within DUWS. There are no evidence-based guidelines currently available to dentists for the control of DUWS contamination in Europe. The aim of this study was to investigate the microbial load of water from DUWS, especially the presence of microbial pathogens which can be a source of cross-contamination between patients. Water and tube samples were obtained from 40 dental surgeries in Barcelona. The samples were taken from turbine and from air/water syringe. Twelve of these DUWS were fed with hard water from mains (with chlorine) and 28 dental units with bottle or tank deionized water. Microbial load was ranged from 0 to 3.6 × 103 colony-forming units/mL. Legionella sp., Mycobacterium sp., Pseudomonas aeruginosa and Candida sp. were found in 4, 8, 6, and 2 different surgeries, respectively. Inclusive oral streptococci were found in 2 dental units indicating that there is a back siphonage in the systems analysed.