Free-living amoebae have been found to be a reservoir for various pathogenic bacteria in aquatic environments. For example, the Acanthamoeba genus renders possible the intracellular multiplication of Legionella pneumophila, which is responsible for legionellosis. It consequently matters to quantify Acanthamoeba cells and thereby enhance our assessment of the risk of contamination. The classical microbiological method of quantification relies on amoebae growth and most probable number calculation. We have developed a real-time PCR assay based on a TaqMan probe that hybridizes onto 18S rDNA. This probe is specific to the Acanthamoeba genus. The assay was successful with both the trophozoite and the cyst forms of Acanthamoeba. Highly sensitive, it proved to permit detection of fewer than 10 cells, even those that are not easily cultivable, such as the cyst forms.