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Background Protecting healthcare workers (HCW) from Coronavirus Disease-19 (COVID-19) is critical to preserve the functioning of healthcare systems. We therefore assessed seroprevalence and identified risk factors for Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) seropositivity in this population. Methods Between June 22nd and August 15th 2020, employees from healthcare institutions in Northern/Eastern Switzerland were screened for SARS-CoV-2 antibodies. We recorded baseline characteristics, non-occupational and occupational risk factors. We used pairwise tests of associations and multivariable logistic regression to identify factors associated with seropositivity. Findings Among the 4664 included HCW from 23 healthcare facilities, 139 (3%) were seropositive. Non-occupational exposures independently associated with seropositivity were contact with a COVID-19 positive household (adjusted OR=54, 95%-CI: 31-97) and stay in a COVID 19 hotspot (aOR=2.2, 95%-CI: 1.1-3.9). Blood group 0 vs. non-0 (aOR=0.4, 95%-CI: 0.3-0.7), active smoking (aOR=0.5, 95%-CI: 0.3-0.9) and living with children <12 years (aOR=0.3, 95%-CI: 0.2-0.6) were associated with decreased risk. Occupational risk factors were close contact to COVID-19 patients (aOR=2.8, 95%-CI: 1.5-5.5), exposure to COVID-19 positive co-workers (aOR=2.0, 95%-CI: 1.2-3.1), poor knowledge of standard hygiene precautions (aOR=2.0, 95%-CI: 1.3-3.2), and frequent visits to the hospital canteen (aOR=1.9, 95%-CI: 1.2-3.1). Interpretation We identified several modifiable factors associated with SARS-CoV-2 seropositivity among our HCW. Living with COVID-19 positive households showed by far the strongest association. The lower risk among those living with children, even after correction for multiple confounders, is remarkable and merits further study. Funding Swiss National Sciences Foundation, Federal Office of Public Health, Health Department Canton of St. Gallen