The aim of this cross-sectional study is to address whether health literacy (HL) and vaccine confidence are related with influenza vaccination uptake among staff of nursing homes (NHs). It was conducted in Tuscany (Italy) in autumn 2018, including the staff of 28 NHs. A questionnaire was used to collect individual data regarding influenza vaccination in 2016–2017 and 2017–2018 seasons; the intention to be vaccinated in 2018–2019; as well as demographic, educational, and health information. It included also the Italian Medical Term Recognition (IMETER) test to measure HL and eight Likert-type statements to calculate a Vaccine Confidence Index (VCI). The number of employees that fulfilled the questionnaire was 710. The percentage of influenza vaccination uptake was low: only 9.6% got vaccinated in 2016–2017 and 2017–2018 and intended to vaccinate in 2018–2019. The VCI score and the IMETER-adjusted scores were weakly correlated (Rho = 0.156). At the multinomial logistic regression analysis, the VCI was a positive predictor of vaccination uptake. In conclusion, vaccine confidence is the strongest predictor of influenza vaccination uptake among the staff of NHs. The development of an adequate vaccine literacy measurement tool could be useful to understand whether skills could be related to vaccine confidence.