The aim of this study was to assess how well the guidelines on vaccination against hepatitis B had been implemented among healthcare workers (HCWs) at risk for blood exposure. A point-prevalence survey was conducted in six departments of a university hospital in Sweden: the emergency room, intensive care unit, postoperative unit, surgical theatre, department of anaesthesiology and the laboratory for blood chemistry. All HCWs who worked in these departments during the 24h of the survey were asked to complete a questionnaire. In total, 369 questionnaires were analysed. Seventy-nine percent (293/369) of HCWs had received at least one dose of vaccine, but only 40% (147/369) reported that they were fully vaccinated and 21% (76/369) had not been vaccinated at all. The majority of unvaccinated HCWs (72/76, 95%) stated that they would accept vaccination if offered. The main barrier to better compliance with the guidelines is not lack of acceptance among the employees but the failure of the employer to ensure that policies are implemented.