Building on the job demands-resources (JD-R) and the allostatic load (AL) models, in this study we investigated the role of smart working (SW) in the longitudinal association between workload/job autonomy (JA) and a possible biomarker of work-related stress (WRS) in the hair, namely the cortisol to dehydroepiandrosterone sulphate (DHEA(S)) ratio, during the COVID-19 pandemic. Overall, 124 workers completed a self-report questionnaire (i.e., psychological data) at Time 1 (T1) and collected a strand of hair (i.e., biological data) three months later (Time 2, T2). Results from moderated multiple regression analysis showed that smart working at T1 was negatively associated with hair cortisol/DHEA(S) ratio at T2. Additionally, the interaction between workload and SW was significant, with workload at T1 being positively associated with hair cortisol/DHEA(S) ratio at T2 among smart workers. Overall, this study indicates that SW can be conceived as a double-edged sword, with both positive and negative consequences on employee well-being. Furthermore, our findings suggest that hair cortisol/DHEA(S) ratio is a promising biomarker of WRS. Practical implications that organizations and practitioners can adopt to promote organizational well-being are discussed.