Abstract Background We aimed to estimate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence and describe its determinants and associated symptoms among unvaccinated healthcare workers (HCWs) after the first wave of the pandemic. Methods HCWs from 13 Dutch hospitals were screened for antibodies against the spike protein of SARS-CoV-2 in June-July 2020 and after three months. Participants completed a retrospective questionnaire on determinants for occupational and community exposure to SARS-CoV-2 and symptoms suggestive of COVID-19 experienced since January 2020. The seroprevalence was calculated per baseline characteristic and symptom at baseline and after follow-up. Adjusted odds ratios (aOR) for seropositivity were determined using logistic regression. Results Among 2328 HCWs, 323 (13.9%) were seropositive at enrolment, 49 of whom (15%) reported no previous symptoms suggestive of COVID-19. During follow-up, only 1% of the tested participants seroconverted. Seroprevalence was higher in younger HCWs compared to the mid-age category (aOR 1.53, 95% CI 1.07–2.18). Nurses (aOR 2.21, 95% CI 1.34–3.64) and administrative staff (aOR 1.87, 95% CI 1.02–3.43) had a higher seroprevalence than physicians. The highest seroprevalence was observed in HCWs in the emergency department (ED) (aOR 1.79, 95% CI 1.10–2.91), the lowest in HCWs in the intensive, high, or medium care units (aOR 0.47, 95% CI 0.31–0.71). Chronic respiratory disease, smoking, and having a dog were independently associated with a lower seroprevalence, while HCWs with diabetes mellitus had a higher seroprevalence. In a multivariable model containing all self-reported symptoms since January 2020, altered smell and taste, fever, general malaise/fatigue, and muscle aches were positively associated with developing antibodies, while sore throat and chills were negatively associated. Conclusions The SARS-CoV-2 seroprevalence in unvaccinated HCWs of 13 Dutch hospitals was 14% in June-July 2020 and remained stable after three months. A ...