Free-living amoebae (FLA) are widely distributed in the environment and are known causative agents of rare but often serious diseases. Acanthamoebae can cause a rapidly progressing keratitis (AK) especially in contact lens wearers. Moreover, acanthamoebae can cause opportunistic infections, such as granulomatous amoebic encephalitis (GAE). The GAE caused by Balamuthia mandrillaris and the fatal primary amoebic meningoencephalitis (PAME) caused by Naegleria fowleri can both affect also healthy humans. In addition to their role as pathogens, FLA also serve as a reservoir for various bacteria, such as Legionella pneumophila, which increase their infectivity through intracellular replication. Industrial waters and, in particular, cooling towers have been the source of Legionella outbreaks in the past and are therefore subject to periodic screenings, not considering, however, FLA and their reservoir function. Therefore, the aim of the first study was to screen industrial waters for FLA by culture, PCR and sequencing in parallel to a standard investigation on legionellae (ÖNORM ISO 11731:1998). A total of 201 samples were investigated, including 129 cooling waters, 72 process waters and, in addition, 30 cooling lubricants from Austrian paper plants. Altogether, FLA were detected in 72.6% of the water samples, with acanthamoebae being most prevalent (in 23.9% of all samples). Moreover, 34.8% of the water samples were positive for Legionella spp., of which 81.4% were positive for FLA. The cooling lubricants were negative for legionellae and only one sample was positive for Acanthamoeba. Based on these results, the aim of the second study was to incorporate a screening system for host amoebae into a Legionella routine screening. A new real-time PCR screening system specific for different groups of FLA was established for this purpose. Three cooling towers were regularly screened over the period of one year and investigated by culture and molecular biological methods in parallel. Additionally, 12 tap water samples were included in the study. The Legionella screening was performed according to the standard protocol ISO 11731-2:2004. Altogether, 83.3% of the cooling tower samples and 50% of the tap water samples were positive for FLA, and again Acanthamoeba was the most common genus. It is noteworthy that, due to the high organic burden, 69.7% of the cooling tower samples were unsuitable for the standard Legionella screening. In the remaining samples, positivity for Legionella spp. was 25% by culture, but overall positivity was 50% by molecular biological methods, demonstrating a need for improvement of the currently established screening methods. In the last study, we aimed to give an overview of the cases of Acanthamoeba infections diagnosed in Austria during the past 20 years. All samples of patients with suspected AK or GAE were screened for Acanthamoeba spp. by culture, PCR or both, and the detected amoebae were genotyped. A total of 154 cases of AK and three cases of GAE were diagnosed, involving various Acanthamoeba genotypes, but with the most common genotype being T4.