The human pathogenic Legionella bacteria are found ubiquitously in natural and human-made aquatic environments as residents in biofilms, where close interactions with other microorganisms like protozoa are possible. Nosocomial legionellosis already has been linked frequently to Legionella-contaminated artificial water supplies. For this reason, a rapid and accurate detection and quantification of these bacteria in environmental and clinical samples, combined with more information about their behavior in complex microbial communities and diverse ecosystems, is of importance. More insight into the ecology of the Legionella bacteria can lead to new methods to suppress their high numbers in human-made aquatic systems. Fluorescent in situ hybridization (FISH), based on ribosomal ribonucleic acid-targeted oligonucleotide probes, combines the precision and specificity of a molecular technique with the power to visualize individual cells without prior cultivation. In this chapter, the use of FISH for the detection and quantification of Legionella in water samples and in the visualization of these bacteria inside protozoa and biofilms is described in detail.