Examine the associations of occupational and leisure-time physical activity with job stress, burnout, and well-being among healthcare industry workers. Quantitative; cross-sectional. Healthcare Industry. US Amazon Mechanical Turk participants (n = 550) employed in the healthcare industry, worked 35 hours or more per week, had ≥ 1 supervisor and ≥ 1 co-worker, and were ≥ 18 years old. Self-reported measures of occupational physical activity (OPA) and leisure-time physical activity (LTPA), employee well-being, job stress, and burnout operationalized as exhaustion and disengagement. Associations between OPA and LTPA with employee well-being, job stress, exhaustion and disengagement were assessed with separate multiple linear regression models. OPA had positive significant associations with job stress (β = 0.10, P value = .003) and exhaustion (β = 0.21, P value < .0001). No significant associations were found between OPA with other psychological outcomes. A significant inverse association was found between LTPA and exhaustion (β = -0.04, P value = .007). In a sample of U.S. health care workers, and consistent with prior epidemiological studies, greater LTPA was associated with lower feelings of exhaustion. In contrast, health care workers with greater OPA reported higher perceptions of job stress and exhaustion. The findings underscore the need for more research aimed at understanding relationships between OPA and psychological health among healthcare workers.