The aim of this study was to identify the most effective disinfection protocol to reduce the presence of Legionella pneumophila in the water system of two Italian hospitals. From 2004 to 2009, 271 samplings of hot water were carried out in 11 hospital units to detect the presence of L. pneumophila. Additionally, water samples collected from one boiler outlet and the hot water recirculation were tested. From 2004 to 2009, L. pneumophila was present in 37% of the samples. Of these, 68.3% and 18.8% were positive for serogroups 2-14 and 1, respectively. Furthermore, 12.9% of the samples were positive for both serogroups. Finally, a maximal count of 10(4) CFU/L was measured in the most distal sites. To reduce L. pneumophila colonization, a two-year long hyperchlorination (2004-2006) was carried out. Moreover, from June 2005 until now, continuous maintenance of boilers and tanks, substitution of the shower heads and increase of the boiler outlet temperature to 60 °C were performed. All these treatments led to a marked reduction of L. pneumophila colonization in the short but not in the medium-long term. Only the use of chlorine dioxide led, after four years, to a reduction of the loads of L. pneumophila to values below 100 CFU/L. However, in the distal sites a persistent degree of colonization (maximum value 700 CFU/L, average 600 CFU/L) was observed probably due to the presence of L. pneumophila in the stagnant water in dead legs. In conclusion, data show that long-term chlorination of hot water sources together with carefully aimed maintenance of water pipes can lead to an effective reduction of L. pneumophila concentration in hospital water systems.