Introduction The COVID-19 pandemic has compounded the global crisis of stress and burnout among healthcare workers. But few studies have empirically examined the factors driving these outcomes in Africa. Our study examined associations between perceived preparedness to respond to the COVID-19 pandemic and healthcare worker stress and burnout and identified potential mediating factors among healthcare workers in Ghana. Methods Healthcare workers in Ghana completed a cross-sectional self-administered online survey from April to May 2020; 414 and 409 completed stress and burnout questions, respectively. Perceived preparedness, stress, and burnout were measured using validated psychosocial scales. We assessed associations using linear regressions with robust standard errors. Results 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, healthcare workers 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 to 17% of the effect. Conclusions Low perceived preparedness to respond to COVID-19 increases stress and burnout, and this is partly through fear of infection. Interventions, incentives, and health systemic changes to increase healthcare workers’ morale and capacity to respond to the pandemic are needed.