Nontuberculous mycobacteria (NTM), ubiquitous in water environments, are increasingly recognized as nosocomial pathogens. Our study reports a one-year survey of the water system of two hospitals, A and B, in a small town near Florence, Italy. NTM were found throughout the study period in both settings, but B showed a significantly higher mycobacterial load. Mycobacterium gordonae and Mycobacterium fortuitum were the most frequent species isolated. Identification was carried out by conventional techniques and by high performance liquid chromatography (HPLC) analysis of cell wall mycolic acids. HPLC profiling could be used as a first-choice method for identification of environmental mycobacteria.