Diabetes and cardiovascular diseases are major causes of morbidity and mortality worldwide, and are associated with changes in the human gut microbiota. To better understand the relationships between diet, disease, and the colonic microbiome, we used the in vitro GIS1 system and repetitive element palindromic polymerase chain reaction (rep-PCR) to determine the microbial fingerprints in individuals with these diseases and compared them with the fingerprints in healthy controls. Clear differences were apparent in the three groups. The diabetes group showed significantly increased aerobic bacteria, increased coliforms, and reduced bifidobacteria; the balance between beneficial and pathogenic bacteria was disturbed; significant numbers of clostridia were present; and the proportions of various major bacterial groups were unstable through the length of the colon. The microbiota of the cardiovascular group had high numbers of beneficial strains and more closely resembled the control microbiota. Different patterns of lactic acid bacteria were observed in the three groups, and there was a direct link between the presence of lactate and the colonic pH. Ammonium, a microbial metabolite associated with colonic cancer, was associated with consistently high levels of Gram-positive bacteria in the diabetic patients. In the cardiovascular patients and controls, each colonic segment showed a distinct microbial fingerprint, whereas in the diabetics, the same rep-PCR profile occurred in all three segments. The diversity of beneficial bacteria was reduced in patients with a nutritional or cardiovascular disease. Both diabetes and cardiovascular disease are associated with changes in the colonic microbial fingerprint. This study of microbial microbiota fingerprint modification has direct applicability in medical practice.