Introduction Treatment of infections that require high‐level isolation can cause anxiety and fear among health care workers. Adequate and complete multi‐professional simulation‐based training could reduce those feelings and improve patient care. Objective The purpose of this study was to assess the impact of multi‐professional simulation‐based training on the risk perception and preparedness of health care workers (registered nurses, doctors and ancillary staff) who care for patients assessed to be at risk or confirmed to have Ebola, level 3–4 biohazard. Methods A prospective before–after study was designed. Health care workers who participated in a multi‐professional simulation training course to improve the care of patients potentially infected with Level 3 and 4 biohazards were evaluated about their risk perception. The training was based on clinical scenarios. The evaluation was conducted using questionnaire based on Likert scale. After the training, a satisfaction survey about the most important aspects of the course was also conducted. Results Fifty‐eight health care workers participated in the training course, 22 of whom were registered nurses. Participants presented positive changes after the training, increasing their sense of security, predisposition and confidence (p < 0.000001 for all). Conclusion Multi‐professional simulation‐based training significantly improves the perception of safety and preparedness of health care workers regarding the care of patients potentially infected with Ebola virus and other Level 3–4 biohazards. Relevance to clinical practice The implementation of educational training strategies – such as simulations – is beneficial in improving the capacity of response and coping, as well as in reducing feelings of fear and insecurity.