It is incontrovertible that relationships exist between the intestinal microbiome and intestinal structure, function, health and disease. Understanding these relationships has been enabled through the evolution of microbiology techniques; from growth and enumeration using media selective for specific species to current exploitation of technologies that allow insight regarding the dialogue between complex microbial communities and host responses. In the context of inflammatory bowel disease, most initial microbiology studies involved analysis of stool or excreted mucus samples, in essence reflective of luminal contents transiting the intestinal environment. Endoscopy, biopsies and availability of resected intestinal tissues further enhanced the analysis. However, the majority of studies were observational and, in many cases, attempted to determine cause or effect interactions between predetermined species, mainly bacteria, inflammation or beneficial/probiotic characteristics. Exceptions to this approach included use of denaturing gradient or temperature gradient electrophoresis and shotgun sequencing approaches that facilitated characterisation of subsets of microbial communities. Notably, these advances explored the intestinal mesentery rarely and, when performed, investigations concentrated on adipose components and, as with prior studies on luminal contents or mucus-adhering microflora, failed to determine whether the presence of bacteria was due to passive or active translocation processes or whether detected microbial profiles were unambiguously characteristic of either Crohn’s disease (CD) or ulcerative colitis (UC). We have employed next-generation sequencing of bacteria from mesenteric lymph nodes of IBD patients to clarify these questions somewhat by differentiating CD and UC unequivocally. We have also employed more sophisticated techniques to elucidate the metagenome of mesenteric lymph nodes and confirmed that the bacterial profiles of CD and UC are discrete and distinctive. Further, we have explored emerging data regarding metagenomic innovations and believe that these bacterial signatures, perhaps combined with complementary assays of systemic biomarkers, may allow resolution of the issue of unclassified IBD (IBDU) and selection of optimal therapeutic and/or surgical approaches.