Building on the job demands–resources (JD-R) and allostatic load (AL) models, in the present study we examined 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–dehydroepiandrosterone sulfate (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 provided a strand of hair (i.e., biological data) three months later (Time 2, T2). Results from moderated multiple regression analysis showed that SW at T1 was negatively associated with the hair cortisol/DHEA(S) ratio at T2. Additionally, the interaction between workload and SW was significant, with workload at T1 being positively associated with the hair cortisol/DHEA(S) ratio at T2 among smart workers. Overall, this study indicates that SW is a double-edged sword, with both positive and negative consequences on employee wellbeing. Furthermore, our findings suggest that the hair cortisol/DHEA(S) ratio is a promising biomarker of WRS. Practical implications that organizations and practitioners can adopt to prevent WRS and promote organizational wellbeing are discussed.