Introduction Coronavirus disease 2019 (COVID-19) has been associated with mental health outcomes and healthcare workers (HCWs) are at the highest risk. The aim of this study was to determine the prevalence and predictors of depression, anxiety and stress, among frontline HCWs at COVID-19 isolation and treatment sites in Gaborone, Botswana. Methods This was a cross-sectional study using self-administered questionnaires at the six (6) isolation facilities. The 42-item Depression, Anxiety and Stress Scale (DASS-42) was used to assess for the outcomes. The proportions are presented with 95% confidence intervals (95% CI). Logistic regression analysis identified predictors of the outcomes. A p value of <0.05 was considered significant. Results A total of 447 participants with a median age of 30 years responded. Depression, anxiety and stress were detected in 94 (21.0% (95% CI 17.3–25.1%)), 126 (28.2% (CI 24.1–32.6%)) and 71 (15.9% (12.6–19.6%)) of the participants respectively. Depression was associated with smoking (AOR 2.39 (95% CI 1.23–4.67)), working at the largest COVID-19 isolation centre, Sir Ketumile Masire Teaching Hospital (SKMTH) (AOR 0.25 (95% CI 0.15–0.43)) and experience of stigma (AOR 1.68 (95% CI 1.01–2.81)). Tertiary education (AOR 1.82 (95% CI 1.07–3.07)), SKMTH (AOR 0.49 (95% CI 0.31–0.77)), household members with chronic lung or heart disease (AOR 2.05 (95% CI 1.20–3.50)) and losing relatives or friends to COVID-19 (AOR 1.72 (95% CI 1.10–2.70)) were predictors of anxiety. Finally, predictors of stress were smoking (AOR 3.20 (95% CI 1.42–7.39)), household members with chronic heart or lung disease (AOR 2.44 (95% CI 1.27–4.69)), losing relatives or friends to COVID-19 (AOR 1.90 (1.05–3.43)) and working at SKMTH (AOR 0.24 (0.12–0.49)). Conclusion Depression, anxiety and stress are common among frontline HCWs working in the COVID-19 isolation sites in Gaborone. There is an urgent need to address the mental health outcomes associated with COVID-19 including addressing the risk factors identified in this study.

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