In this research the relationship between hair cortisol and burnout was evaluated through the study of third-variables effect. Hair cortisol concentration was measured in 234 health workers. All participants completed perceived stress survey and Maslach Burnout Inventory. Our results showed that 40% of volunteers presented altered hair cortisol values, 12% burnout, 33% showed high depersonalization, 38% high emotional fatigue, 36% low personal fulfillment and 22% presented 2 of the 3 burnout components. A moderated mediation was found between psychological and biological variables. This research highlights the importance of including a biological variable such as hair cortisol in burnout assessment.