Background Delivery by means of cesarean section has been associated with increased risk of childhood immune-mediated diseases, suggesting a role of early bacterial colonization patterns for immune maturation. Objective We sought to describe the influence of delivery method on gut and airway colonization patterns in the first year of life in the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) birth cohort. Methods Seven hundred children from the COPSAC2010 birth cohort participated in this analysis. Fecal samples were collected at age 1 week, 1 month, and 1 year, and hypopharyngeal aspirates were collected at age 1 week, 1 month, and 3 months and cultured for bacteria. Detailed information on delivery method, intrapartum antibiotics, and lifestyle factors was obtained by personal interviews. Results Seventy-eight percent of the children were born by means of natural delivery, 12% by means of emergency cesarean section, and 9% by means of elective cesarean section. Birth by means of cesarean section was significantly associated with colonization of the intestinal tract by Citrobacter freundii, Clostridium species, Enterobacter cloacae, Enterococcus faecalis, Klebsiella oxytoca, Klebsiella pneumoniae, and Staphylococcus aureus at age 1 week, whereas colonization by Escherichia coli was associated with natural birth. At age 1 month, these differences were less prominent, and at age 1 year, they were not apparent, which was confirmed by means of multivariate data-driven partial least squares analyses. The initial airway microbiota was unaffected by birth method. Conclusion Delivery by means of cesarean section was associated with early colonization patterns of the neonatal gut but not of the airways. The differences normalized within the first year of life. We speculate that microbial derangements, as indicated in our study, can demonstrate a possible link between delivery by means of cesarean section and immune-mediated disease.