During the COVID-19 pandemic, healthcare workers (HCW) have been subjected to greater workloads. We conducted a cross-sectional online survey to assess the impact of the COVID-19 pandemic on Brazilian HCW. Data were collected between 11 August and 1 November 2020. Of the 295 respondents, 95 (32.2%) were medical doctors, 82 (27.8%) administrative staff, 53 (18.0%) nurses, 27 (9.2%) laboratory staff, and 38 (12.9%) were other staff. COVID-19-related restructuring at the health facilities was reported by 207 (70.2%) respondents, and 69 (23.4%) had their tasks changed. Preventive measures were well respected when seeing suspected patients. Overall, 167 (56.6%) HCW screened positive for anxiety and 137 (46.4%) for depression; 109 (36.9%) screened positive for both conditions. Of the 217 (73.6%) HCW who had been tested for COVID-19, at least one positive result was reported in 49 (22.6%). Following a positive COVID-19 test, 45/49 (91.8%) stopped working and stayed home. In conclusion, we found a high incidence of COVID-19 infection among Brazilian HCW with high rates of anxiety and depression despite a good self-reported adherence to COVID-19 preventive measures. As such, our study highlights the urgent need for interventions to mitigate the psychosocial risks HCW in Brazil encounter during the COVID-19 pandemic.