Health workers (HW) are at increased risk for SARS-CoV-2 infection. In order to monitor the infection dynamic on the basis of contact with patients, HW at the St. Antonius Hospital (SAH) were tested four times in one year by PCR and serology. The cumulative incidence of infection in HW was calculated. Swab and blood tests were simultaneously performed between April 2020 and April 2021. Risk factors and demographic information were assessed at the beginning of the study. The response rate was above 75% in all rounds of testing. The study comprised 1506 HW, 165 (10.6%) of which tested positive for SARS-CoV-2 infection. Working in an ICU or on wards with patient contact were risk factors (OR 4.4, 95% CI 1.73–13.6 and OR 2.9, 95% CI 1.27–8.49). At the end of the study, the majority of HW (810 of 1363 (59.4%)) had been vaccinated at least once. A total of 29.1% of unvaccinated HW and 5.3% of vaccinated HW showed an immune response typical for natural SARS-CoV-2 infection. Of the 73 HW who provided information on the course of the disease, 31.5% reported that their quality of life continued to be impaired. The cumulative incidence of infection was low in these HW, which may be attributed to vaccination and good hygiene. Nevertheless, a work-related infection risk was identified, highlighting the need to improve protection against infection. A high risk of developing long COVID was found after the infection has subsided. Special rehabilitation programs should be provided and HW should be compensated for reduced work capacity in the case that rehabilitation fails or takes a long time.