Objectives To describe prevalence and incidence of anti-SARS-CoV-2 antibodies among Belgian hospital healthcare workers (HCW) in April–December 2020. Design Prospective cohort study. Follow-up was originally planned until September and later extended. Setting Multicentre study, 17 hospitals. Participants 50 HCW were randomly selected per hospital. HCW employed beyond the end of the study and whose profession involved contact with patients were eligible. 850 HCW entered the study in April–May 2020, 673 HCW (79%) attended the September visit and 308 (36%) the December visit. Outcome measures A semiquantitative ELISA was used to detect IgG against SARS-CoV-2 in serum (Euroimmun) at 10 time points. In seropositive samples, neutralising antibodies were measured using a virus neutralisation test. Real-time reverse transcription PCR (RT-qPCR) was performed to detect SARS-CoV-2 on nasopharyngeal swabs. Participant characteristics and the presence of symptoms were collected via an online questionnaire. Results Among all participants, 80% were women, 60% nurses and 21% physicians. Median age was 40 years. The seroprevalence remained relatively stable from April (7.7% (95% CI: 4.8% to 12.1%) to September (8.2% (95% CI: 5.7% to 11.6%)) and increased thereafter, reaching 19.7% (95% CI: 12.0% to 30.6%) in December 2020. 76 of 778 initially seronegative participants seroconverted during the follow-up (incidence: 205/1000 person-years). Among all seropositive individuals, 118/148 (80%) had a positive neutralisation test, 83/147 (56%) presented or reported a positive RT-qPCR, and 130/147 (88%) reported COVID-19-compatible symptoms at least once. However, only 46/73 (63%) of the seroconverters presented COVID-19-compatible symptoms in the month prior to seroconversion. Conclusions The seroprevalence among hospital HCW was slightly higher than that of the general Belgian population but followed a similar evolution, suggesting that infection prevention and control measures were effective and should be strictly maintained. After two ...