Objectives: Burnout is a stress-related, psychological syndrome due to high levels of job stressors. It has been found to be related to impairments of well-being, health, and job outcomes. Alterations of glucocorticoid secretion might be a mechanism explaining the linkage between burnout and reduced psychophysical functioning. Regarding hair cortisol as indicator this assumption, so far, has been only examined in cross-sectional studies. Therefore, we aimed to compare cross-sectional and prospective associations between different burnout symptoms and hair cortisol, additionally investigating potential nonlinear associations. Methods: The prospective study sample comprises 194 employees (95% nurses) from German geriatric care. We assessed burnout symptoms at baseline (t1) and 6 months later (t2) and collected hair samples for cortisol analyses at t2. Results: We found significant cross-sectional and prospective nonlinear (i.e., exponential) but not linear relationships between an aggregated measure of the burnout subscales emotional exhaustion, cynicism, and reduced efficacy and hair cortisol, even after adjusting for BMI and depressive mood. None of the single subscales of burnout was related to hair cortisol after adjusting for confounders. Conclusions: Our findings further support the assumption that accumulated burnout symptoms and hypercorticolism are positively related.