OBJECTIVE: Identification of hospital preparedness challenges against biological events such as coronavirus disease 2019 (COVID-19) is essential to improve dynamics, quality, and business continuity confidence in the health system. Accordingly, the purpose of the present study is to evaluate the challenges of hospital preparedness in biological events. METHODS: This study used a qualitative method using content analysis in which 20 health-care managers and experts who are experienced in biological events were selected through purposeful sampling. The data collection was done through semi-structured interviews, which continued until data saturation. The data were analyzed using qualitative content analysis as well as the Landman and Graneheim Approach. RESULTS: Six main concepts (training and practice, resource management, safety and health, patient management, risk communication, and laboratory and surveillance) and 14 subconcepts were extracted on hospital preparedness challenges in biological events through analyzing interviews. CONCLUSIONS: The present study indicated that the health system of the country faces many challenges in response to biological events and threats. Moreover, study participants indicated that Iranian hospitals were not prepared for biological events. It is recommended to design preparedness plans of hospitals based on preparedness standards for biological events. In addition, comprehensive measures are required to enhance their capacity to respond to biological emergencies.