Background: Healthcare workers are continuously exposed to a high level of stress, especially emergency department professionals. In the present research, we aimed to determine the internal consistency and validity of the Stress Factors and Manifestations Scale for in-hospital and out-of-hospital emergency workers. Methods: A quantitative, prospective, cross-sectional, and observational study including 269 emergency service professionals. Results: The scale was composed of 21 items, with a Cronbach’s α value of 0.908. The hospital workers (38.4 ± 10.8 vs. 35.1 ± 9.9, p = 0.014) and women (39.3 ± 11.4 vs. 34.2 ± 8.6, p < 0.001) had higher levels of stress. The sensitivity, specificity, and predictive values of the scale were adequate. Conclusion: In the present study, including in-hospital and out-of-hospital emergency workers, the Stress Factors and Manifestations Scale presented appropriate usefulness, internal consistency, and validity, with optimal predictive ability. Higher levels of anxiety, female gender, being less optimistic, and working in hospital emergency departments were related to increased stress levels. Further studies are warranted to validate our results and potentially extend the Stress Factors and Manifestations Scale to other contexts.