Active shooter incidents represent an increasing threat to American society, especially in commercial and educational buildings. In recent years, a wide variety of security countermeasures have been recommended by public and governmental agencies. Many of these countermeasures are aimed to increase building security, yet their impact on human behavior when an active shooter incident occurs remains underexplored. To fill this research gap, we conducted virtual experiments to evaluate the impact of countermeasures on human behavior during active shooter incidents. A total of 162 office workers and middle/high school teachers were recruited to respond to an active shooter incident in virtual office and school buildings with or without the implementation of multiple countermeasures. The experiment results showed countermeasures significantly influenced participants’ response time and decisions (e.g., run, hide, fight). Participants’ responses and perceptions of the active shooter incident were also contingent on their daily roles, as well as building and social contexts. Teachers had more concerns for occupants’ safety than office workers. Moreover, teachers had more positive perceptions of occupants in the school, whereas office workers had more positive perceptions of occupants in the office.