Vaccination to prevent influenza virus infection and to lessen its severity is recommended among healthcare workers (HCWs). Health professionals have a higher risk of exposure to viruses and could transmit the influenza virus to vulnerable patients who are prone to severe disease and mortality. The aim of the current study was to evaluate the levels of influenza vaccine acceptance and uptake as well as its determinants, among Jordanian HCWs over the last influenza season of 2021/2022. This study was based on a self-administered electronic survey that was distributed in March 2022. Psychological determinants of influenza vaccine acceptance and vaccine conspiracy beliefs were assessed using the previously validated 5C scale questionnaire (confidence, complacency, constraints, calculation and collective responsibility) and the vaccine conspiracy beliefs scale. The study sample comprised a total of 1218 HCWs: nurses (n = 412, 33.8%), physicians (n = 367, 30.1%), medical technicians (n = 182, 14.9%), pharmacists (n = 161, 13.2%) and dentists (n = 87, 7.1%), among others. About two-thirds of the study sample expressed willingness to receive influenza vaccination if provided free of charge (n = 807, 66.3%), whereas less than one-third were willing to pay for the vaccine (n = 388, 31.9%). The self-reported uptake of the influenza vaccine in the last influenza season was 62.8%. The following factors were significantly associated with higher acceptance of influenza vaccination if provided freely, as opposed to vaccine hesitancy/rejection: male sex; physicians and dentists among HCW categories; higher confidence and collective responsibility; and lower complacency, constraints and calculation. Higher influenza vaccine uptake was significantly correlated with nurses and physicians among HCW categories, older age, a higher monthly income, higher confidence and collective responsibility, lower complacency and constraints and lower embrace of general vaccine conspiracy beliefs. The results of the current study can provide helpful clues to improve influenza vaccine coverage among HCWs in Jordan. Consequently, this can help to protect vulnerable patient groups and reserve valuable resources in healthcare settings. Psychological determinants appeared to be the most significant factors for vaccine acceptance and uptake, whereas the embrace of general vaccine conspiracy beliefs was associated with lower rates of influenza vaccine uptake, which should be considered in educational and interventional measures aiming to promote influenza vaccination.