In 2005, WHO developed the campaign "Clean care is safer care", aiming at the reduction of Healthcare Associated Infections (HCAI) through the worldwide promotion of appropriate hand hygiene practices. Adherence to these practices presently ranges from 5 to 81% (average 40%). Aim of the study was the evaluation of healthcare workers (HCWs) adherence to the application of WHO guidelines on the hand hygiene and the possible impact on HCAI reduction. First, some wards at highest HCAI risk were identified. Then, direct observational survey was conducted to evaluate the behaviour of the different HCW categories. Six-hundred-twenty-eight HCWs were observed: 519 nurses (68 in training), 109 physicians (63 in training). Survey analysis highlighted that compliance with handwashing was higher in post care practices (59.6%) than before (55.1%). Some HCWs performed handwashing in both cases. Only in 30.6% of cases HCWs observed the handwashing technique suggested by the guidelines. In 70.4% they wore gloves during procedures in which it was possible to have contact with potentially infectious material, but they did not change them before caring for a new patient in 64% of cases. The survey confirms the international data, showing a low compliance with hand hygiene practices by the HCWs and the need to promote a correct handwashing based on respect of the international guidelines.