Healthcare workers (HCWs) are an important group of professionals exposed to biological risk during their work activities. So, the aim of this study is to perform a survey on the knowledge, attitude and behaviour of Italian HCWs towards the vaccinations recommended by the Ministry of Health. A cross-sectional study was carried out during the period September 2014–August 2015 in the Lazio region. The study was conducted by recruiting HCWs and biomedical students. The sample was comprised of 571 responders, of whom 12.4% were physicians, 18.9% were nurses, 34.3% were other HCW, and 34.3% were biomedical students (medical and nurses students). Hepatitis B virus (HBV) is perceived as a risk for personal health by 457 (80%) participants; TB is also worrying (434; 76%). Moreover, HBV (70.9%) and tuberculosis (TB) (79.2%) are perceived as a risk for health, while influenza is not considered so by most participants (46.2%). There is an underestimation of the role of influenza, perceived as a risk for 137 respondents (24%). The vaccination rate among these HCWs is highest for Hepatitis B virus (HBV) (82%), and lowest for influenza (28.5%) and varicella (40.3%). The vast majority of responders are in favour of HBV (77.8%) and TB (64.8%) vaccines. For other vaccinations there is less interest (between 33% and 40% for measles, mumps, rubella, pertussis and influenza). This study shows that knowledge of recommended occupational vaccinations is insufficient in HCWs, with few exceptions represented by HBV and TB. There is a need for novel approaches in this field, with the aim of enhancing vaccine coverage among HCW.