Human microbiome research has been enabled and empowered by the rapid advances in DNA/RNA sequencing technologies, and provided new insights into the potential roles for the microbiome in health and disease. There has also been a shift in approach, with shotgun metagenome sequencing (MGS) now being adopted to produce an unbiased functional assessment of the microbiota, in comparison to the taxonomic profiling afforded by 16S rRNA gene-amplicon sequencing. However, while MGS is rapidly becoming the preferred approach with samples that are rich in microbial biomass (e.g. stool) constraints remain with the effective use of MGS with DNA from samples with limited microbial density and/or rich in non-microbial DNA. A specific example are the mucosa-associated microbiota (MAM) that reside along the human gastrointestinal tract, which changes in its density, form, and function relative to anatomical site, gender, and/or health status. Here, we will present our development and evaluation of microbe-culture metagenome sequencing (MC-MGS) to better characterise the MAM of Crohn’s disease (CD) patients. In this approach, total DNA is extracted from biopsy samples, and a subsample is subjected to the subtractive removal of human DNA using the NEBNEXT protocol; which results in a 2-8 fold enrichment of microbial DNA, as assessed by qPCR. A second matched biopsy that has been cryopreserved in anaerobic buffer is used to inoculate a habitat simulating medium, to produce a MAM-derived consortia and from which metagenomic DNA is extracted and sequenced. Our preliminary results show that the outgrowth cultures show PCR-positivity for fastidious anaerobes such as Faecalibacterium prausnitzii. Additionally, we have been able to isolate urease-producing facultative anaerobes from some biopsies. These findings suggest that MC-MGS can provide a useful, additive approach to produce a more detailed, comprehensive and functional understanding of the MAM in patients with CD, and other chronic, non-communicable diseases.