A metabolic disorder such as Type 2 Diabetes mellitus (T2DM) is a complex disease induced by genetic, environmental and nutritional factors. The db/db mouse model, bearing a non-functional leptin receptor is widely used to investigate the pathophysiology of T2DM. Fecal extracts of db/db and wild type littermates were studied to unravel a broad spectrum of new and relevant metabolites related to T2DM as proxies of the interplay of gut microbiome and murine metabolomes. The non-targeted metabolomics approach consists of an integrated analytical concept of high-resolution mass spectrometry FT-ICR-MS followed by UPLC-TOF-MS/MS experiments. We demonstrate that a metabolic disorder such as T2DM affects the gastrointestinal tract environment thereby influencing different metabolic pathways and their respective metabolites in diabetic mice. Fatty acids, bile acids concerning cholic and deoxycholic acid and steroid metabolism were highly discriminative comparing fecal metabolomes of wt and db/db mice. Furthermore, sulfur-(S)-containing metabolites including N-acyl taurines were altered in diabetic mice, enabling to focus on S-containing metabolites, especially the sulfate and taurine conjugates of bile and fatty acids. Different sulfate containing bile acids including sulfocholic acid, oxocholic acid sulfate, taurocholic acid sulfate and cyprinol sulfate were significantly altered in diabetic mice. Moreover, we identified twelve new sulfate and taurine conjugates of hydroxylated fatty acids with significant importance in T2DM metabolism in db/db mice.