Diagnostic approaches to COVID-19 include clinical history, PCR tests for the presence of SARS-CoV-2 virus and detection of antibodies. By combining these three approaches, the seroprevalence of anti–SARSCoV- 2 antibodies can be examined in healthcare teams. The aim of the study was to examine the seroprevalence of anti–SARSCoV- 2 antibodies in a population of healthcare professionals 6–8 weeks after the first COVID-19 case was detected in the Czech Republic. A total of 269 subjects were enrolled in the study (187 women, 82 men) with a median age of 45.9 years (21 – 71 years). We used a questionnaire to ascertain travel history and clinical signs of any respiratory tract infection. Blood samples were collected, and IgG levels were analysed in all samples. The level of IgA antibodies was analysed in those positive for IgG. PCR testing was performed in cases testing positive for presence of antibodies. The enzyme-linked immunosorbent assay (ELISA) test system for SARS-CoV-2 from Euroimmun (Germany) was used to analyse immunoglobulin levels. 17% of the tested cohort reported symptoms compatible with COVID-19 and 35.8% reported history of international travel. There were 5 subjects positive IgG cases (of 269; 1.85%), and one IgA positive and IgG borderline positive subject (0.37%). There was only one PCR positive subject. Anti SARS-CoV-2 antibodies were thus detected in 2.22% of participating health professionals. This article shows the pitfalls of the testing methods and highlights the necessity of using a correct testing algorithm, considering the character of the tested population and the expected low prevalence. © 2020, Czech Medical Association J.E. Purkyne. All rights reserved.