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Objectives: This study investigated working environment factors associated with healthcare workers’ job stress in a public medical hospital during COVID-19. Methods: We surveyed healthcare workers in a public medical center in September of 2020. Multivariable linear regression analysis was performed to find significant factors associated with increased job stress of healthcare workers. Results: Increased musculoskeletal pain, difficulties in intra-departmental communication, difficulties in co-work with other departments, and insufficient personal protective equipment significantly was associated with increased job stress while coping with COVID-19. When musculoskeletal pain increased, having difficulties in co-work with other departments was associated with increased job stress. Conclusions: Increased job stress was associated with increased musculoskeletal pain, difficulties in intra-departmental communication, difficulties in co-work with other departments, and insufficient personal protective equipment.