Background and objectives: Burnout is a syndrome typically occurring in work environments with continuous and chronic stress. Physicians are at increased risk for burnout, as a result of 24-h work, delayed work–life balance gratification, and the challenges associated with patient care. The aim of the present study was to evaluate the psychological parameters of burnout symptoms in relation to biomarkers of stress among physicians with different medical specialties. Materials and methods: A total of 303 physicians were contacted as potential participants. A comparison group of 111 individuals working outside medicine was used as a control to verify the results. The physicians were specialists in internal medicine, general surgery, pathology, and primary care. Serum cortisol, salivary cortisol, adrenocorticotropic hormone (ACTH), insulin (IRI), and prolactin levels were analyzed by chemiluminescence enzyme immunoassay (Access 2, Beckman Coulter). Fasting glucose in serum and glycated hemoglobin (HbA1C) in whole blood were measured using the automatic analyzer AU 480 Beckman Coulter system. Symptoms of burnout were measured with the Maslach Burnout Inventory (MBI). Results: The group with burnout presented significantly higher levels of serum and saliva cortisol, ACTH, prolactin, fasting glucose, and HbA1C compared with the control group. The correlation analysis between biomarkers showed a positive correlation with moderate strength between serum and saliva cortisol (r = 0.516, p = 0.01),as well as serum and saliva cortisol with ACTH (r = 0.418; r = 0.412, p = 0.01) and HbA1C (r = 0.382; r = 0.395, p = 0.01). A weak positive correlation was found between serum and saliva cortisol with prolactin (r = 0.236; r = 0.267, p < 0.01) and glucose (r = 0.271; r = 0.297, p < 0.01). In the multiple logistic regression model, saliva cortisol, HbA1C, and age were significantly associated with burnout (chi-square = 16.848, p < 0.032). Conclusion: Our findings demonstrated the interest of exploring biomarkers of stress related to burnout in health professionals.