Background Healthcare workers caring for coronavirus disease 2019 (COVID-19) patients are at an increased risk for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The aim of this seroepidemiological study was to evaluate the risk of infection for employees at a tertiary care hospital. Methods Serological tests for antibodies against SARS-CoV-2 were carried out in a prospective cohort of employees directly involved in the care of COVID-19 patients every 2 weeks from March to July 2020 (1st wave). Antibody status was examined again between December 2020 and February 2021 (2nd wave). Results The seroprevalence of antibodies against SARS-CoV-2 was 5.1% at the end of the study in February 2021. The cumulative incidence was 3.9% after a median observation period of 261 days. Conclusion We observed a low risk of SARS-CoV-2 infection comparable to that of the general population in the examined cohort of healthcare workers involved in the acute care of COVID-19 patients under the applied hygiene and protective measures.