Police officers are often required to perform under high-stress circumstances, in which optimal task performance is crucial for their and the bystanders’ physical integrity. However, stress responses, particularly anxiety and increased cortisol levels, shift attention from goal-directed to stimulus-driven control, leaving police officers with poor shooting performance under stress. Cardiac vagal activity and coping-related traits (i.e., self-control, sensation seeking) might help individuals to maintain performance under stress. So far, only few studies have integrated coping-related traits, psychophysiological stress markers and occupationally meaningful measures of behavior to investigate police officers’ work performance under stress. Therefore, the present study investigated 19 police recruits (
M
age
= 22.84,
SD
= 3.30) undergoing a reality-based shooting scenario in two experimental conditions in a within-design: low stress (LS) against a non-threatening mannequin, and high stress (HS), involving physical threat by an opponent. Psychological (i.e., anxiety, mental effort) and physiological stress responses (i.e., salivary cortisol, alpha-amylase, cardiac vagal activity) as well as shooting accuracy were repeatedly assessed. It was hypothesized that under stress, police recruits would demonstrate elevated psychophysiological stress responses and impaired shooting performance. Elevated psychophysiological stress responses would negatively influence shooting performance, whereas self-control, sensation seeking and cardiac vagal activity would positively influence shooting performance. While recruits reported significantly higher anxiety and mental effort in the HS scenario, both scenarios elicited comparable physiological responses. Overall, shooting accuracy was low and did not significantly decrease in the HS scenario. Shooting performance was predicted by self-control in the LS scenario and by post-task cardiac vagal activity in the HS scenario. While increased anxiety hints at a successful stress manipulation, physiological responses suggest similar stress levels for both scenarios, diminishing potential behavioral differences between the scenarios. Performance efficiency decreased under stress, as indicated by increasing mental effort. Findings on self-control suggest that suppressing negative stress responses might lead to impaired goal-directed attention, resulting in performance decrements. For police research and training, high-realism scenarios afford an opportunity to investigate and experience psychophysiological stress responses.