BackgroundAppropriate hand hygiene behavior is much crucial during COVID-19 pandemic, which is influenced by workload, mental health and professional quality of life. The increasing workload, worse mental health and negative professional quality of life are severe challenges for healthcare workers, whereas the evidence of mechanism between them and hand hygiene behavior are limited. This study aimed to understand and model how HCWs’ hand hygiene behaviors were influenced by workload, mental health and professional quality of life.MethodsA total of 1,734 healthcare workers were investigated, covering self-reported hand hygiene behavior, mental health, workload, professional quality of life and socio-demographic characteristics. Structural equation modelling was conducted to confirm interrelationships and parameters among the variables.Findings The final model had a good fit (CFI=0.980; TLI=0.941; RMSEA=0.055). Higher workload was linked to worse mental health (β = 0.165, p < 0.001) and higher burnout (β = 0.183, p < 0.001). Worse mental health was linked to higher burnout (β = 0.339, p < 0.001). The higher burnout was associated with worse self-reported hand hygiene behavior (β = -0.302, p < 0.001).ConclusionWorkload is positively associated with mental health and burnout; mental health is positively related to burnout; burnout is negatively associated with hand hygiene behavior. Thus, hand hygiene behavior can be promoted by the strategy to decrease the workload, burnout and enhance mental health.