The contribution of sustainably food processing to healthy intestinal microbial functions is of recent acquisition. The sourdough fermentation fits well with the most sustainable bread making. We manufactured baker's yeast (BYB) and sourdough (t-SB30) breads, which first underwent to an in-depth characterization. According to nutritional questionnaires, we selected 40 volunteers adhering to the Mediterranean diet. Data on their fecal microbiota and metabolome allowed the selection of two highly representative fecal donors to separately run the Twin Mucosal-SHIME (Twin M-SHIME) under 2-week feeding with BYB and t-SB30. Bread feeding did not affect the microbial composition at phylum and family levels of both donors, in all Twin M-SHIME colon tracts, and lumen and mucosal compartments. The genus core microbiota showed few significant fluctuations, which regarded the relative abundances of Lactobacillus and Leuconostoc according to feeding with BYB and t-SB30, respectively. Compared with BYB, the content of all short chain fatty acids (SCFA), and isovaleric and 2-methylbutyric acids significantly increased with t-SB30 feeding. This was evident for all Twin M-SHIME colon tracts and both donors. The same was found for the content of Asp, Thr, Glu, GABA, and Orn. The bread characterization made possible to identify the main features responsible for this metabolic response. Compared with BYB, t-SB30 had much higher contents of resistant starch, peptides, and free amino acids, and an inhomogeneous microstructure. We used the most efficient approach to investigate a staple food component, excluding interferences from other dietary factors and attenuating human physiology overlaps. The daily consumption of sourdough bread may promote the healthy microbiota metabolism at colon level. Knowledge on environmental factors, which may compose the gut microbiota, and drive the host physiology and health is of paramount importance. Human dietary habits and food compositions are pivotal drivers to assemble the human gut microbiota, but, inevitably, unmapped for many diet components, which are poorly investigated individually. Approximately 30% of the human diet consists of fermented foods and beverages. Bread, a fermented/leavened food, is a basic component of the human diet. Its potential effect on gut microbiota composition and functionality is challenging. In this study, we industrially made baker's yeast and sourdough breads, which were used to feed the Twin Mucosal-SHIME, a worldwide scientifically validated gastrointestinal simulator. Only the consumption of sourdough bread has the potential to enhance the synthesis of short chain fatty acids and free amino acids at the colon level.