ABSTRACT The multiplication of opportunistic pathogens in drinking water supplies might pose a threat to public health. In this study, distributed unchlorinated drinking water from eight treatment plants in the Netherlands was sampled and analyzed for fungi, nontuberculous mycobacteria (NTM), and several opportunistic pathogens by using selective quantitative PCR methods. Fungi and NTM were detected in all drinking water samples, whereas Legionella pneumophila , Pseudomonas aeruginosa , Stenotrophomonas maltophilia , and Aspergillus fumigatus were sporadically observed. Mycobacterium avium complex and Acanthamoeba spp. were not detected. Season had no influence on the occurrence of these organisms, except for NTM and S. maltophilia , which were present in higher numbers in the summer. Opportunistic pathogens were more often observed in premise plumbing water samples than in samples from the distribution system. The lowest number of these organisms was observed in the finished water at the plant. Thus, fungi, NTM, and some of the studied opportunistic pathogens can multiply in the distribution and premise plumbing systems. Assimilable organic carbon (AOC) and/or total organic carbon (TOC) had no clear effects on fungal and NTM numbers or on P. aeruginosa - and S. maltophilia -positive samples. However, L. pneumophila was detected more often in water with AOC concentrations above 10 μg C liter −1 than in water with AOC levels below 5 μg C liter −1 . Finally, samples that contained L. pneumophila , P. aeruginosa , or S. maltophilia were more frequently positive for a second opportunistic pathogen, which shows that certain drinking water types and/or sampling locations promote the growth of multiple opportunistic pathogens.