Influenza is an infectious disease with a high impact on the population in terms of morbidity and mortality, but despite International and European guidelines, vaccination coverage rates among healthcare workers (HCWs) remain very low. The aim of the present study was to evaluate influenza vaccination adherence in the three Sicilian University Hospitals of Catania, Messina, and Palermo and to understand the attitudes and perceptions of vaccinated healthcare workers and the main reasons for vaccination refusal. A cross-sectional survey through a self-administered questionnaire was conducted during the 2019/2020 influenza season. Overall, 2356 vaccinated healthcare workers answered the questionnaire. The main reason reported for influenza vaccination adherence during the 2019/2020 season was to protect patients. Higher self-perceived risk of contracting influenza and a positive attitude to recommending vaccination to patients were significantly associated with influenza vaccination adherence during the last five seasons via multivariable analysis. Fear of an adverse reaction was the main reason for influenza vaccine refusal. In accordance with these findings, Public Health institutions should develop and tailor formative and informative campaigns to reduce principal barriers to the immunization process and promote influenza vaccination adherence among HCWs.