Background: Coronavirus disease (COVID-19) serology testing evaluates the prevalence of COVID-19 cases. Methods: A seroepidemiological survey of COVID-19 among healthcare workers was performed (June 2020 to November 2020) in Ribeirão Preto, São Paulo, Brazil. Overall, 10,172 and 2,129 workers participated in the first and second phases, respectively. Results: First phase: 12.7% tested positive for COVID-19 (73.5% females and 35.2% aged 30-39 years), and 29.6% were nursing technicians. Second phase: 12.1% tested positive for COVID-19 (65.5% females and 33.3% aged 40-49 years), and 24.8% were nursing assistants. Conclusions: In 2020, healthcare workers in Ribeirão Preto had COVID-19 in a similar way.