- BACKGROUND: Early antibiotic exposure is linked to persistent disruption of the infant gut microbiome and subsequent elevated pediatric asthma risk. Breastfeeding acts as a primary modulator of the gut microbiome during early life, but its effect on asthma development has remained unclear. METHODS: We harnessed the CHILD cohort to interrogate the influence of breastfeeding on antibiotic-associated asthma risk in a subset of children (n = 2,521). We then profiled the infant microbiomes in a subset of these children (n = 1,338) using shotgun metagenomic sequencing and compared human milk oligosaccharide and fatty acid composition from paired maternal human milk samples for 561 of these infants. FINDINGS: Children who took antibiotics without breastfeeding had 3-fold higher asthma odds, whereas there was no such association in children who received antibiotics while breastfeeding. This benefit was associated with widespread "re-balancing" of taxonomic and functional components of the infant microbiome. Functional changes associated with asthma protection were linked to enriched Bifidobacterium longum subsp. infantis colonization. Network analysis identified a selection of fucosylated human milk oligosaccharides in paired maternal samples that were positively associated with B. infantis and these broader functional changes. CONCLUSIONS: Our data suggest that breastfeeding and antibiotics have opposing effects on the infant microbiome and that breastfeeding enrichment of B. infantis is associated with reduced antibiotic-associated asthma risk. FUNDING: This work was supported in part by the Canadian Institutes of Health Research; the Allergy, Genes and Environment Network of Centres of Excellence; Genome Canada; and Genome British Columbia.