Objective To study the SARS-CoV-2 infection rate among hospital healthcare workers after the first wave of the COVID-19 pandemic, and provide more knowledge in the understanding of the relationship between infection, symptomatology and source of infection. Design A cross-sectional study in healthcare workers. Setting Northern Limburg, the Netherlands. Participants All employees of VieCuri Medical Center (n=3300) were invited to enrol in current study. In total 2507 healthcare workers participated. Intervention Between 22 June 2020 and 3 July 2020, participants provided venous blood samples voluntarily, which were tested for SARS-CoV-2 antibodies with the Wantai SARS-CoV-2 Ig total ELISA test. Work characteristics, exposure risks and prior symptoms consistent with COVID-19 were gathered through a survey. Main outcome measure Proportion of healthcare workers with positive SARS-CoV-2 serology. Results The overall seroprevalence was 21.1% (n=530/2507). Healthcare workers between 17 and 30 years were more likely to have SARS-CoV-2 antibodies compared with participants >30 years. The probability of having SARS-CoV-2 antibodies was comparable for healthcare workers with and without direct patient (OR 1.42, 95% CI 0.86 to 2.34) and COVID-19 patient contact (OR 1.62, 95% CI 0.80 to 3.33). On the contrary, exposure to COVID-19 positive coworkers (OR 1.83, 95% CI 1.15 to 2.93) and household members (OR 6.09, 95% CI 2.23 to 16.64) was associated with seropositivity. Of those healthcare workers with SARS-CoV-2 antibodies, 16% (n=85/530) had not experienced any prior COVID-19-related symptoms. Only fever and anosmia were associated with seropositivity (OR 1.90, 95% CI 1.42 to 2.55 and OR 10.51, 95% CI 7.86 to 14.07). Conclusions Healthcare workers caring for hospitalised COVID-19 patients were not at an increased risk of infection, most likely as a result of taking standard infection control measures into consideration. These data show that compliance with infection control measures is essential to control secondary ...