Aims: To confirm the presence of viable Legionella spp. in dental unit water-lines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. Methods and Results: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two- and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. Conclusions: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. Significance and Impact of the Study: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.