Teachers are among the occupational groups with the highest sick leave rates due to workplace stress and burnout symptoms. A substantial body of research has suggested social isolation and neuroticism to be related to physiological stress activity. However, the relationship between such characteristics and stress experiences has rarely been studied in conjunction with physiological stress indicators in the teachers’ natural settings. Thus, the present study examines salivary cortisol and α-amylase as physiological stress indicators on teachers’ work and leisure days and their relationship with social isolation. Furthermore, we test whether neuroticism moderates the relationship between social isolation and salivary biomarkers. Forty-two teachers completed questionnaires assessing social isolation (Trier Inventory for the Assessment of Chronic Stress) and neuroticism (Big-Five Inventory). Participants collected eight saliva samples on three days, two workdays, and one leisure day to measure the concentration of cortisol and α-amylase as biomarkers of the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS), respectively. Results showed a significantly higher Cortisol Awakening Response (CAR) and diurnal cortisol slope (DCS) on workdays than on the leisure day but no significant differences regarding measures of α-amylase. We found a significant positive relationship between social isolation and the CAR on the leisure day but no association with the α-amylase measures. Furthermore, after controlling for confounders, social isolation was unrelated to neuroticism, and the latter did not moderate between social isolation and the CAR. Our findings suggest an association between social isolation and the HPA axis, i.e., the CAR, but do not support an association with the ANS, which would be indicated by the α-amylase assessments. Finally, our findings could not support an association of neuroticism with the HPA axis and ANS.