Lesbian, gay, and bisexual (LGB) individuals report higher levels of stressful interpersonal conflicts with others because of their divergence from heterosexual social norms. In a biopsychosocial model of minority stress, we tested diurnal cortisol slopes and internalized homonegativity (IH) as two potential mechanisms linking experiences of LGB-related stress to depression. The sample consisted of 27 lesbian and bisexual young women and 35 gay and bisexual young men ( N = 62; age, 17–27, M = 21.34 years) from the greater metropolitan area of Montréal, Québec. We predicted that (a) LGB-related stress, IH, and diurnal cortisol slopes would be positively associated with each other and with depression; and (b) flatter diurnal cortisol slopes and/or greater IH would mediate the link between LGB-related stress and depression. LGB-related stress, diurnal cortisol slopes, and IH were positively associated with depression, and mediation analyses showed that diurnal cortisol slopes mediated the link between LGB-related stress and depression. These findings suggest that external stressors associated with being LGB can impact individuals’ physiological coping resources, thus affecting their psychological health.