Healthcare-associated infections (HAI) are the most frequent and severe complication acquired in healthcare settings and have a significant impact in terms of morbidity, mortality and costs. This document is aimed at different health professionals and focuses on the role of molecular epidemiology in the prevention and management of these infections. It describes the role of molecular characterization and of bioinformatics archives, the organizational levels of laboratories, the evidence regarding cost-effectiveness, ethical aspects related to HAI, and highlights some specific peculiarities of Italy. Molecular epidemiology is an indispensable tool and should be part of a multidisciplinary approach in the proper management of HAI.