The main objective of the study is assessing knowledge, opinions and compliance with the procedures of health professionals (physician, nurses, medical and nursing students) about Hand Hygiene (HH). There is a number of research which indicates that physicians respect less than nurses Hand Hygiene, there are a smaller number which investigates the differences in the attitude of the aforementioned subjects during their studies. A questionnaire was administered to a sample of 756 participants (252 doctors, 268 nurses, 117 nursing students and 119 students of medicine) at a large University Hospital in Rome, to determine their knowledge, compliance and procedures with Hand Hygiene. Knowledge of medical students is lower than that of nursing students, as well as they have lower values in adherence to practice. In both HH Beliefs Scale (HBS) and HH Practices Inventory (HHPI) questionnaires, nursing students have higher scores than nurses, doctors and medical students. The questions on the use of alcohol-based cleaners have been those where there was the lowest number of correct responses, across all professions. Physicians compared to nurses have a lower adherence to Hand Hygiene. Future research should clarify what the differences are in the construction of the study on the practice of washing hands, what are the barriers to health professionals and best methods for teaching habits, namely the effectiveness of hand washing. Professionals should be more involved in the fight against Healthcare-associated infections.