Background: Job stress threatens teachers’ well-being and health. Identifying physiological factors that underline job stress is crucial for teachers’ health and students’ learning. This cross sectional study examined the cycle of stress biomarkers (salivary cortisol and alpha amylase) over the course of teaching among Iranian English teachers. Materials and Methods: 59 English teachers from two foreign language institutes in Bushehr province, southern Iran volunteered to participate in this study. The participants’ saliva samples were collected three times over the course of a usual teaching day (before class, during class, and after class). Salivary alpha amylase and cortisol levels were analyzed in the biomarker Sina Lab in Bushehr using commercially available and research-based kinetic reaction (sAA) (Pars Test) and immunoassay (cortisol) kits (IBL). Results: A significant pattern was found for alpha amylase while cortisol did not show any significant change over the course of teaching. Conclusion: The findings highlighted the usefulness and importance of measuring physiological biomarkers in studying teachers’ stress