The first months of life are critical for microbiome acquisition and mark the earliest interactions between the colonising microbiota and maturing immune system. The lung harbours a variety of microorganisms thought to be critical in contributing to physiological homeostasis and protecting against opportunistic pathogens. Although metagenomic sequencing technologies continue to uncover the diverse microbial community in the human respiratory tract, isolate-level resolution through bacterial culturing and functional analysis of the microbiota across body sites remains limited. Through the GLAM&I (Gut, Lung and their Microbiomes & Immunology) clinical study, longitudinal and time-matched respiratory and stool samples have been collected from 108 neonatal and paediatric patients at Monash Children’s Hospital. Bacterial culturing protocols have been optimised for culture of diverse respiratory bacteria and used to purify 5,480 bacterial isolates from 208 respiratory samples within the cohort. Equivalent analysis of gastrointestinal microbes using established protocols identified 1,657 bacterial isolates from 62 stools. To evaluate microbial community composition, respiratory and stool samples were subjected to shotgun metagenomic sequencing. Measures of alpha diversity show changes in species richness and evenness in the respiratory tract and gut over the first weeks of life, as well as patient specific compositional differences between these two sites. The combination of culturing and shotgun metagenomic analysis highlights site-specific bacterial isolate colonisation and the dominance of Firmicutes during early life. Furthermore, 30 bacterial species were demonstrated to be shared across the respiratory tract and gut microbiomes in early life. The application of sophisticated culturing technologies to the respiratory microbiome provides the opportunity to advance beyond microbiome associations to isolate-level causative validation. Specific individual bacterial isolates purified through this work will enable validation the site-specific functional roles and immune interaction during early life. A deeper functional understanding of the microbiota and their interactions with the host will be critical in guiding the development of novel therapeutics and microbiome-based medicines.