The current study explored how religiosity and spirituality may differentially influence substance use by sexual identity based on a sample of adult sexual minority women (SMW; n = 437 lesbian; n = 323 bisexual) relative to a heterosexual comparison sample (n = 636). We examined three questions: (1) whether spirituality was differentially associated with alcohol and marijuana use by sexual identity; (2) whether religiosity was differentially associated with alcohol and marijuana use by sexual identity; (3) whether observed differences between spirituality or religiosity and substance use by sexual identity persisted after adjusting for religious environment. Measures included spirituality (importance of spirituality), religiosity (importance of religion, attending religious services), and past year substance use (alcohol use disorder [AUD], any marijuana use, and regular marijuana use). Higher levels of spirituality were associated with increased odds of AUD among both lesbian and bisexual respondents relative to heterosexuals. Higher levels of religiosity among lesbian participants were associated with increased odds of AUD relative to heterosexuals with higher levels of religiosity. Consistent with theories of minority stress, findings suggest that spirituality and religiosity are less protective for SMW than heterosexual women and, in some cases, may contribute to greater risk of substance use.