Challenging interactions are the main source of teacher’ stress in the classroom. We investigated the association of chronic stress and characteristics of teacher-student interactions with teachers’ Hair Cortisol Concentration (HCC). Forty-one teachers (27 women; Mage = 39.65 ± 12.14 years; Mlesson number = 23.15 ± 3.99 lessons per week; grade: elementary, secondary, high, and vocational school teachers) participated in the present study, with participation lasting over the length of one year. HCC was assessed from a 3 cm hair segment near the scalp. Self-reported chronic stress in the last three months was further assessed using the ‘Trier Inventory for Chronic Stress’ (TICS). Additionally, four consecutive, same-day lectures of each teacher were videotaped and coded offline in an event sampling procedure by trained external observers. The videos were analyzed for two stressors, i.e., classroom disruptions and total student aggression, as well as two resources, i.e., teacher-student relationship and classroom management. Overall, hair samples were collected M = 120.34 days (SD = 84.39) after the distribution of the questionnaires, and M = 67.63 days (SD = 18.40) prior to the observations. Lesson number, classroom disruptions, as well as total student aggression were all significantly positively correlated with HCC. In addition, both teacher-student relationship and classroom management were significantly negatively related to HCC. With regard to self-rated chronic stress, only the TICS subscale ‘Pressure to perform’ was positively related to HCC. Exploratory moderation analyses revealed that an increasingly good, observed teacher-student relationship buffered the positive association between lesson number and HCC. Our findings show significant associations between HCC and mainly objectively assessable stress, supporting HCC as a biological indicator of chronic stress. In this association, a good relationship between teachers and students acts as a buffer. While the findings underline the importance of examining objective and behavioral data for better understanding the psychobiology of stress, they also support the importance of boostering teachers’ (social) resources to increase their overall resilience.