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Early childhood care and education (ECCE) supports children’s development. ECCE teachers, however, incur stress and negative outcomes (e.g., health, classroom relationships/environment). Investigations examining teacher stress typically implement psychological measures. Yet, these reflect one aspect of stress. Physiological measures (e.g., hair cortisol concentration; HCC) are complementary. The current study, grounded in Ecological Systems Theory and the Transactional Model of Stress and Coping, examined (1) ECCE teachers’ perceived working conditions, stress, and self-efficacy, and (2) whether working conditions and self-efficacy were associated with perceived stress or HCC. Psychological stress results were moderate; better working conditions, pay/promotion opportunities, and self-efficacy were each significantly and positively associated with lower perceived stress. Physiological results trended toward better working conditions and self-efficacy associated with lower HCC.