The Co-HCW study is a prospective cohort study among hospital staff, including healthcare workers (HCWs) and administration staff, at the Jena University Hospital (JUH), Germany. The objectives of this study were to assess SARS-CoV-2 IgG seroprevalence, individual exposure risk factors and compliance of HCWs to wear personal protective equipment (PPE). After the first nosocomial COVID-19 outbreak at JUH, mandatory masking was implemented on 20th March 2020. We evaluated point seroprevalence using two IgG detecting immunoassays and issued a questionnaire to assess COVID-19 exposure, clinical symptoms and compliance to wear PPE. Antibody retesting was offered to participants with a divergent result of both immunoassays 5-10 weeks after the first test. Between 19th May and 19th June 2020, we analysed 660 participants [out of 3,228; 20.4%]. Among them, 212 participants (32.1%) had received a previous COVID-19 test. Four of them (1.9%) reported a positive test result. After recruitment, 18 participants (2.7%) had SARS-CoV-2 antibodies in at least one immunoassay. Overall, 21 participants (3.2%) had any evidence of a past or current SARS-CoV-2 infection. Among them, 13 (61.9%) were not aware of direct COVID-19 exposure and 9 (42.9%) did not report any clinical symptoms. COVID-19 exposure at home (adjusted OR (aOR) with 95% CI: 47.82 (5.49, 416.62)) was associated with SARS-CoV-2 seroprevalence. We observed no evidence for an association between seroprevalence and exposure at work (aOR 0.48 (0.13, 1.70)) or with COVID-19 risk area according to the working place (aOR for intermediate-risk vs. high-risk: 1.97 (0.42, 9.22), aOR for low-risk versus high-risk: 2.10 (0.40, 11.06); p = .655). Reported compliance of HCWs to wear PPE differed (p < .001) between working in high-risk (98.3%) and in intermediate-risk areas (69.8%). In conclusion, compared to administration staff, we observed no additional risk to acquire SARS-CoV-2 infections by patient care, probably due to high compliance to wear PPE.