BACKGROUND: The study examined the association between prenatal tobacco or co-exposure to tobacco and cannabis and childrens cortisol reactivity at kindergarten age and the role of child sex, maternal negative mood (depression/perceived stress), and parenting behavior during play interactions as moderators of this association. METHODS: The sample was 238 mother-child dyads (67 tobacco users, 83 co-users of tobacco and cannabis, and 88 non-users). Data used were obtained from pregnancy assessments and six postnatal assessments at 2, 9, 16, 24, and 36 months and kindergarten age. Infant cortisol was measured in response to two laboratory stress paradigms. RESULTS: Co-exposed children had a significantly greater decrease from pre-stressor to post-stressor and overall lower cortisol response compared with non-exposed children. This association was moderated by maternal harshness during play interactions across early childhood. Co-exposed children had flatter cortisol responses regardless of the mothers level of harshness or stress/depression. However, non-exposed children who experienced low harshness had the normative cortisol peak 20 min post-stressor, while non-exposed children with high maternal harshness had a flatter cortisol pattern. Similarly, non-exposed children with more depressed/stressed mothers had higher pre-stressor cortisol levels, while those who experienced low maternal depression/stress had lower pre-stressor cortisol but peaked post-stress. CONCLUSIONS: Results suggest that prenatal polysubstance exposure is associated with greater risk for lower cortisol response in children and highlight the role of parenting behavior for non-exposed but not the co-exposed children.