Vaccination of healthcare workers (HCWs) is a crucial element to overcome the COVID-19 pandemic. The aim of this survey was to assess attitudes, sources of information and practices among Italian Healthcare workers (HCWs) in relation to COVID-19 vaccination. Methods: From 19 February to 23 April 2021, an anonymous voluntary questionnaire was sent to the mailing list of the main National Health Service structures. Data were collected through the SurveyMonkey platform. Results: A total of 2137 HCWs answered. Hesitancy towards COVID-19 vaccination was more frequent in females, in those with lower concern about COVID-19, and in nurses, auxiliary nurses (AN) and healthcare assistants. Hesitant professionals were more likely to not recommend vaccination to their patients or relatives, while a high concern about COVID-19 was related to an increased rate of recommendation to family members. HCWs were mostly in favor of mandatory vaccination (61.22%). Female sex, a lower education level, greater hesitancy and refusal to adhere to flu vaccination campaigns were predictors influencing the aversion to mandatory vaccination. All categories of HCWs referred mainly to institutional sources of information, while scientific literature was more used by professionals working in the northern regions of Italy and in infection control, infectious diseases, emergencies and critical areas. HCWs working in south-central regions, nurses, AN, healthcare technicians, administrators and HCWs with a lower education level were more likely to rely on internet, television, newspapers, and the opinions of family and friends. Conclusions: Communication in support of COVID-19 immunization campaigns should consider the differences between the various HCWs professional categories in order to efficiently reach all professionals, including the most hesitant ones.