The aim of the present study was to determine the prevalence of Legionella spp. in water systems of hospitals and hotels located in South Western Greece, to study the molecular epidemiology of the isolated strains and their possible association with bacterial contamination (total count and Pseudomonas aeruginosa), the water pH, and temperature. A prevalence survey for Legionella spp. by culturing techniques in water distribution systems of eight hospitals and nine hotels occurred in South Western Greece. Water sampling and microbiological analysis were carried out following the ISO methods. Legionella pneumophila was detected in 33% and 36% of the distribution systems of hospitals and hotels, respectively. Our survey results suggest a frequent prevalence of elevated concentrations of Legionella spp. in water systems of hospitals and hotels. Our investigation has confirmed the need to regularly monitor the microbiological condition of water systems in hospitals and hotels.