Background: The COVID-19 pandemic has compounded the global crisis of stress and burnout among healthcare workers (HCWs). But few studies have empirically examined the factors driving these outcomes. We examined the association between perceived preparedness to respond to the pandemic and HCW stress and burnout and identified potential mediating factors among HCWs in Ghana.Methods: Data are from HCWs in Ghana who completed a cross-sectional self-administered online survey; 414 and 409 HCWs completed stress and burnout questions, respectively. Perceived preparedness, stress, and burnout were measured using validated psychosocial measures. We assessed associations using linear regressions with robust standard errors.Findings: The average score for preparedness was 24 (SD=8.8), 16.3 (SD=5.9) for stress, and 37.4 (SD=15.5) for burnout. In multivariate analysis, HCWs who felt somewhat prepared and prepared had lower stress (β=-1.89, 95%CI:-3.49 to -0.30 and β=-2.66, 95%CI:-4.48 to -0.84) and burnout (β=-7.74, 95%CI:-11.8 to -3.64 and β=-9.25, 95%CI:-14.1 to –4.41) scores than those who did not feel prepared. Appreciation from management and family support were associated with lower stress and burnout, while fear of infection was associated with higher stress and burnout. Fear of infection partially mediated the relationship between perceived preparedness and stress/burnout, accounting for about 16 to17% of the effect.Interpretation: Low perceived preparedness to respond to COVID-19 increases stress and burnout, and this is partly through fear of infection. Interventions to increase HCWs’ morale and capacity to respond to the pandemic are needed.Funding Statement: The UCSF COVID-19 Related Rapid Research Pilot Initiative (#2016796).Declaration of Interests: All authors declare that they have no conflict of interest.Ethics Approval Statement: Ethical approval was obtained from the University of California, San Francisco (#20-30656) and the Navrongo Health Research Centre (#NHRCIRB374).