Background The COVID-19 pandemic has put chronic pressure on worldwide healthcare systems. While the literature regarding the prevalence of psychological distress and associated risk factors among healthcare workers facing COVID-19 has exploded, biological variables have been mostly overlooked. Methods 467 healthcare workers from Quebec, Canada, answered an electronic survey covering various risk factors and mental health outcomes three months after the onset of the COVID-19 pandemic. Of them, 372 (80%) provided a hair sample, providing a history of cortisol secretion for the three months preceding and following the pandemic's start. We used multivariable regression models and a receiver operating characteristic curve to study hair cortisol as a predictor of burnout and psychological health, together with individual, occupational, social, and organizational factors. Results As expected, hair cortisol levels increased after the start of the pandemic, with a median relative change of 29% ( IQR = 3–59%, p < 0.0001). There was a significant association between burnout status and change in cortisol, with participants in the second quarter of change having lower odds of burnout. No association was found between cortisol change and post-traumatic stress disorder, anxiety, and depression symptoms. Adding cortisol to individual-occupational-socio-organizational factors noticeably enhanced our burnout logistic regression model’s predictability. Conclusion Change in hair cortisol levels predicted burnout at three months in health personnel at the onset of the COVID-19 pandemic. This non-invasive biological marker of the stress response could be used in further clinical or research initiatives to screen high-risk individuals to prevent and control burnout in health personnel facing an important stressor.