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This study examines stress biomarkers over the process of teaching and their associations with demographics, emotions, and mental health of 56 English as Foreign Language teachers. Participating teachers were invited to complete a questionnaire packet and provide saliva samples over three-time points (i.e., before, during, and end of teaching). Afterward, the saliva samples were assayed for cortisol and [alpha]-amylase as stress biomarkers. The results showed a significant change in the concentrations of cortisol and [alpha]-amylase over the teaching process. In addition, being female, younger, more educated, and having more teaching experience were significantly associated with a higher level of [alpha]-amylase, particularly before and during class time, thereby reflecting a greater level of stress. However, emotions and general mental health were not significantly associated with cortisol and [alpha]-amylase over time. The findings highlighted the changes of biomarkers over the process of teaching and their relationships with other variables.