Background: Gender differences in vaccine acceptance among health care workers (HCWs) are well documented, but the extent to which these depend on occupational group membership is less well studied. We aimed to determine vaccine acceptance and reasons of hesitancy among HCWs of respiratory clinics in Germany with respect to gender and occupational group membership. Methods: An online questionnaire for hospital staff of all professional groups was created to assess experiences with and attitudes towards COVID-19 and the available vaccines. Employees of five clinics were surveyed from 15 to 28 March 2021. Results: 962 employees (565 [72%] female) took part in the survey. Overall vaccination acceptance was 72.8%. Nurses and physicians showed greater willingness to be vaccinated than members of other professions (72.8%, 84.5%, 65.8%, respectively; p = 0.006). In multivariate analyses, worries about COVID-19 late effects (odds ratio (OR) 2.86; p < 0.001) and affiliation with physicians (OR 2.20; p = 0.025) were independently associated with the willingness for vaccination, whereas age <35 years (OR 0.61; p = 0.022) and worries about late effects of vaccination (OR 0.13; p < 0.001) predicted vaccination hesitancy; no differences were seen with respect to gender. In separate analyses for men and women, only for men worries about COVID-19 late effects were relevant, while among women, age <35 years, worries about late effects of vaccination and worries about COVID-19 late effects played a role. Conclusions: There was no overall difference in vaccination acceptance between male and female HCWs, but there were gender-specific differences in the individual reasons on which this decision-making was based.