Microscopic examination of samples from an algal mat biofilm community in Nymph Creek, Yellowstone National Park, WY, showed the presence of free-living, vahlkampfiid amoebae. Sequence analysis of portions of 18S rRNA genes from a community DNA clone library revealed, among other sequences, several that matched Vahlkampfia. Because some vahlkampfiids, including the pathogens Acanthamoeba and Naegleria fowleri, can cause severe and often fatal infections in humans, we developed a rapid method for sampling and analyzing the mat community using primers targeting Naegleria species. We identified sequences that closely match Acanthamoeba sp. and N. fowleri, as well as Naegleria sequence types not previously described, in Nymph Creek and a variety of other aquatic geothermal sites in Yellowstone and Grand Teton National Parks. Since a number of bacteria, including the intracellular human pathogen Legionella pneumophila, are known to survive in microbial biofilms and as endosymbionts in free-living amoebae, we used traditional culture-based methods in addition to PCR-amplification with primer sets that target 16S rRNA genes, to identify five Legionella species in Nymph Creek. In addition, one potentially new Legionella species was identified in a Euglena enrichment culture obtained from Nymph Creek.