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 these processes. Although metagenomic sequencing technologies continue to detect the diverse microbial community in the human respiratory tract, isolate-level resolution through bacterial culturing and functional analysis of the microbiota remains limited. To evaluate microbial communities, longitudinal and time-matched respiratory (n=208) and stool (n=62) samples have been collected from 157 neonatal patients at Monash Children’s Hospital and subjected to shotgun metagenomic sequencing and isolate culturing. Alpha diversity measures 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. Bacterial culturing protocols were optimised for culture of diverse respiratory bacteria and used to purify 5,480 bacterial isolates from respiratory samples. Equivalent analysis of gastrointestinal microbes, using established protocols, identified 1,657 bacterial isolates from stools, with all cultured bacterial isolates available through the Australian Microbiome Culture Collection (AusMiCC). The combination of culturing and shotgun metagenomic analysis highlights site-specific bacterial isolate colonisation and dominance of the Firmicutes phylum during early life. Specific individual bacterial isolates cultured and 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 is critical in guiding the development of novel therapeutics and microbiome-based medicines.