Emergency medical services (EMS) personnel play an important role in infectious disease control at the forefront of the emergency medical system. However, the increase in EMS personnel’s workload due to the prevalence of infectious diseases can negatively affect the practice of infection control. Accordingly, this study identified the infection control organizational culture, infection control fatigue, and burnout of EMS personnel and analyzed the correlation. In 2023, data on infection control organizational culture, infection control fatigue, and burnout were collected using a structured questionnaire for 104 participating EMS personnel in the Seoul metropolitan area. The findings showed that the personnel were unaware of the infection control organizational culture, and the degrees of infection control fatigue and burnout were high. The more negatively the personnel perceived the infection control organizational culture and the higher the infection control fatigue, the higher the burnout. Therefore, policies to manage EMS personnel’s infection control organizational culture, infection control fatigue, and burnout should be prepared and implemented.