Vaccinations remain the most effective way of preventing infection, disease, and mortality. Public health institutions consequently recommend vaccines to target groups, including healthcare workers, who are considered to be more at risk of exposure and transmission. The aim of this cross-sectional study is to assess, through the administration of a questionnaire, the nursing staff’s knowledge and attitude towards recommended vaccinations, and to explore the effects of a training course (carried out according to the academic detailing methodology) aimed at increasing operators’ knowledge and outreach on recommended vaccinations among healthcare workers. A total of 85 HCWs (30 nursing coordinators and 55 nurses) completed the questionnaire. Results demonstrate a higher rate of agreement towards vaccinations in nursing staff answers (75%), if compared with results of other studies (62–63%). Statistically significant differences between nursing coordinators and nurses can be found. Regarding vaccination attitudes, nursing coordinators agreed in 86% of the answers on healthcare workers’ vaccination vs 70% of nurses (p < 0.001). Considering immunization for influenza, 57% of nursing coordinators vs 18% of nurses reported for vaccination (p < 0.001). Educational programs, carried out according to academic detailing methods, could impact on vaccination attitudes and raise awareness about recommended vaccinations among healthcare workers. The questionnaire is a useful tool for investigating nursing staff knowledge and attitudes towards vaccinations, and to implement strategies to promoting vaccinations among healthcare workers.