Floating biofilms develop at the water-air interface and harbor numerous microorganisms, some of which are human pathogens like Legionella pneumophila. The presence of Legionella spp. and especially L. pneumophila in such biofilms was investigated. In parallel, the occurrence of Naegleria spp., Acanthamoeba spp., Willaertia spp., Vahlkampfia spp. and Hartmanella spp. was determined and it was examined whether Acanthamoeba spp. isolates were naturally infected with L. pneumophila bacteria. Eight anthropogenic and 37 natural aquatic environments were sampled between June and August 2005. Both Legionella spp. and L. pneumophila were present in 100% of the floating biofilms of the anthropogenic aquatic systems. Eighty-one percent of all natural floating biofilm samples were positive for Legionella spp. and 70% of these samples were positive for L. pneumophila. Legionella concentrations were in the range of 10(1)-10(2)cells/cm(2). Naegleria spp. and Acanthamoeba spp., two well-known L. pneumophila amoeba hosts, were present in 50-92% and 67-72% of floating biofilm samples, respectively. Acanthamoeba spp. isolates appeared to be naturally infected with L. pneumophila bacteria as proved by fluorescent in situ hybridization.