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Objectives: This study aimed to investigate the psychological impact of the COVID-19 outbreak on healthcare workers across multiple hospitals in different districts in Botswana. Methods: We conducted a cross-sectional study in five public-funded hospitals from three districts in Botswana from 1 June 2020 to 30 October 2020. We used the neuroticism subscale of the 44-item Big Five Inventory, Patient Health Questionnaire, the Oslo 3-item Social Support Scale, the Anxiety Rating Scale, and the 14-item Resilience Scale to obtain data from 355 healthcare workers. Results: The participants’ mean age (standard deviation) was 33.77 (6.84) years. More females (207, 59%) responded than males (144, 41%). Anxiety and depression were experienced by 14% and 23% of the participants, respectively. After multiple regression analyses, neuroticism predicted depression ( B  = 0.22; p  < 0.01) and anxiety disorder ( B  = 0.31; p  < 0.01). Lower educational status ( B  = −0.13; p  = 0.007) predicted anxiety and younger age ( B  = −0.10; p  = 0.038) predicted depression, while resilience negatively correlated with both disorders. Conclusion: There is a need to develop and implement interventions targeted at these identified risk and protective factors that can be easily delivered to healthcare workers during this pandemic.