Many disinfection treatments can be adopted for controlling opportunistic pathogens in hospital water networks in order to reduce infection risk for immunocompromised patients. Each method has limits and strengths and it could determine modifications on bacterial community. The aim of our investigation was to study under real-life conditions the microbial community associated with different chemical (monochloramine, hydrogen peroxide, chlorine dioxide) and non-chemical (hyperthermia) treatments, continuously applied since many years in four hot water networks of the same hospital. Municipal cold water, untreated secondary, and treated hot water were analysed for microbiome characterization by 16S amplicon sequencing. Cold waters had a common microbial profile at genera level. The hot water bacterial profiles differed according to treatment. Our results confirm the effectiveness of disinfection strategies in our hospital for controlling potential pathogens such as Legionella, as the investigated genera containing opportunistic pathogens were absent or had relative abundances ≤1%, except for non-tuberculous mycobacteria, Sphingomonas, Ochrobactrum and Brevundimonas. Monitoring the microbial complexity of healthcare water networks through 16S amplicon sequencing is an innovative and effective approach useful for Public Health purpose in order to verify possible modifications of microbiota associated with disinfection treatments.