The aims of this study were to identify the best threshold value for the real-time PCR method in detecting the presence of Legionella pneumophila in water samples, and to evaluate the prognostic significance of negative results obtained with the molecular method. From 2011 to 2014, 77 water samples were collected from hospital wards of a large University teaching hospital in Rome (Italy) and screened for L.pneumophila by the standard culture method and by real-time PCR. The high sensitivity and negative predictive value of real-time PCR make this method suitable as a quick screening tool to exclude the presence of L. pneumophila in water samples in the hospital setting.