Background and Objective: Working life is a delicate balance between demands and abilities to meet them. Challenges exceeding individual resources can affect both job performance and the overall workers’ Quality of Life (QoL). Assuming Emotional Exhaustion (EE) acting as a mediator, our aim was to assess the role of Physical Activity (PA), number of diseases, and some work-related factors (workload (WL), Job Insecurity (JI), and autonomy (JA)) in modulating workers’ QoL. Methods: A web survey was administered in July 2021 to 650 Italian workers. A series of regressions were carried out to test the role of EE as mediator between socio-demographic (Sex, Age, n° of diseases), lifestyle (Smoking habit, PA level), and work-related factors (WL, JI, JA, and Classification) and the four domains of QoL defined by the WHO (physical, psychological, social relationships, and environmental). Results: The response rate was 51.1% (21-63 yrs., 66% males, 79% white-collars, 76% providing a complete report). Concerning the physical domain, WL (B=0.42, 95% C.I. (0.331, 0.509), p<0.0001) and medium-to-high PA level (B=−0.11, 95% C.I. (−0.213, −0.012), p=0.029) significantly affected EE, which had a negative relationship with QoL (B=−0.48, 95% C.I. (−0.580, −0.380), p<0.0001). We observed a full mediation for WL while PA still maintained a positive impact on QoL (B=0.11, 95% C.I. (0.023, 0.198), p=0.014). EE did not mediate the negative effect of diseases (B=−0.32, 95% C.I. (−0.400, −0.231), p<0.0001) and JI (B= −0.14, 95% C.I. (−0.232, −0.054), p=0.002) on QoL. PA seemed to not affect the remaining QoL domains. Conclusions: EE is a core component of burnout syndrome, a condition that, although being a widely discussed issue in modern societies, is still not recognised clinically. The suggested possible role of PA in reducing EE and improving workers’ QoL, at least physically, could be worthy of attention in defining strategies to promote workers’ well-being.