This study aimed to investigate the perceived work stress and its influencing factors among hospital staff during the novel coronavirus (COVID‐19) pandemic in Taiwan. A web‐based survey was conducted at one medical center and two regional hospitals in southern Taiwan, targeting physicians, nurses, medical examiners, and administrators. The questionnaire included items on the demographic characteristics of hospital staff and a scale to assess stress among healthcare workers caring for patients with a highly infectious disease. A total of 752 valid questionnaires were collected. The hospital staff reported a moderate level of stress and nurses had a highest level of stress compared to staff in the other three occupational categories. The five highest stress scores were observed for the items “rough and cracked hands due to frequent hand washing and disinfectant use,” “inconvenience in using the toilet at work,” “restrictions on eating and drinking at work,” “fear of transmitting the disease to relatives and friends,” and “fear of being infected with COVID‐19.” Discomfort caused by protective equipment was the major stressor for the participants, followed by burden of caring for patients. Among participants who experienced severe stress (n = 129), work stress was higher among those with rather than without minor children. The present findings may serve as a reference for future monitoring of hospital staff's workload, and may aid the provision of support and interventions.