OBJECTIVES: This study aimed to evaluate whether postpartum maternal stress is associated with infant gastrointestinal microbiome composition and diversity, and whether this relationship may be mediated by maternal caregiving and breastfeeding behaviors and human milk microbiome (HMM) composition. METHODS: Infant fecal and human milk samples were collected from 51 exclusively breastfeeding mother-infant dyads in the Pacific Northwest between 1 and 6 months postpartum. Infant fecal samples with sequencing read counts > 773 (n = 48) and milk samples with read counts > 200 (n = 46) were analyzed for bacterial alpha diversity (richness, Shannon diversity), beta diversity (Bray-Curtis dissimilarity), and genera differential abundances. Infant fecal microbiome (IFM) measures were tested for associations with mothers' self-reported Parenting Stress Index total and subscale scores in regression (richness, Shannon diversity), envfit (beta diversity), and MaAsLin2 (genera abundance) models. Potential mediators of the relationship between maternal stress and IFM were explored (observed total time breastfeeding; maternal-infant physical contact frequency; and HMM alpha diversity, beta diversity, and genera abundance). RESULTS: Maternal stress was not associated with IFM alpha or beta diversities. Two maternal stress subscales were associated with differential abundances of Erysipelotrichaceae UCG-003 (positively) and Eggerthella (negatively) in infant feces. Maternal total stress and two stress subscales (Role Restriction, Attachment) were associated positively with HMM beta diversity (q = 0.07) and negatively with HMM richness (q = 0.08, q = 0.03). CONCLUSIONS: Postpartum stress is not consistently associated with IFM composition during exclusive breastfeeding. However, postpartum maternal stress is associated with HMM diversity, suggesting that maternal stress might influence other developmental pathways in the breastfeeding infant.