Transmission of pathogens via healthcare workers' (HCWs) hands is one of the most frequent means of spreading multi-resistant organisms and occurring healthcare-associated infections (HAIs) in hospitals. The role of contaminated hands in pathogen transmission was recognized by Hungarian physician, Ignác Semmelweis. Hand hygiene prevents cross-infections in hospitals, but numerous epidemiological and microbiology-based studies have documented low compliance of HCWs with this simple procedure. Furthermore, hand hygiene perception of HCWs plays an important role in determining hand hygiene compliance. Our aim was to describe the opinion of HCWs about their perception regarding hand hygiene practice. Our further goal was to strengthen a laboratory basis for bacterial backup control of nosocomial pathogens. A cross-sectional descriptive study was conducted between December 2010 and February 2011 in 13 participating hospitals in Hungary. HCWs know that there is correlation between contaminated hands and HAIs (83%), but neither the frequency (62%) nor the implementation (73%) of their hand hygiene performance are satisfying.We recommend that multimodal interventions - highlighted active microbiological surveillance of HCWs' hands - are the most suitable strategies to reduce the occurrence of HAIs and to determine their impact on cross-transmission of microorganisms and to overcome barriers of HCWs.