(1) Background: early in the COVID-19 pandemic, reverse transcription polymerase chain reaction (RT-PCR) testing was limited. Assessing seroprevalence helps understand prevalence and reinfection risk. However, such data are lacking for the first epidemic wave in Belgian nursing homes. Therefore, we assessed SARS-CoV-2 seroprevalence and cumulative RT-PCR positivity in Belgian nursing homes and evaluated reinfection risk. (2) Methods: we performed a cross-sectional study in nine nursing homes in April and May 2020. Odds ratios (ORs) were calculated to compare the odds of (re)infection between seropositive and seronegative participants. (3) Results: seroprevalence was 21% (95% CI: 18–23): 22% (95% CI: 18–25) in residents and 20% (95% CI: 17–24) in staff. By 20 May 2020, cumulative RT-PCR positivity was 16% (95% CI: 13–21) in residents and 8% (95% CI: 6–12) in staff. ORs for (re)infection in seropositive (compared to seronegative) residents and staff were 0.22 (95% CI: 0.06–0.72) and 3.15 (95% CI: 1.56–6.63), respectively. (4) Conclusion: during the first wave, RT-PCR test programmes underestimated the number of COVID-19 cases. The reinfection rate in residents was 3%, indicating protection, while it was 21% in staff, potentially due to less cautious health behaviour. Future outbreaks should use both RT-PCR and serological testing for complementary insights into transmission dynamics.
