Effects of pectin, inulin, and their combination on the production of microbiota-derived indoles and short-chain fatty acids (SCFAs) from different colon segments were investigated in a batch system inoculated with microbiota from proximal colon (PC) and distal colon (DC) compartments of the Simulator of Human Intestinal Microbial Ecosystem. Bacteria from DC compartment had a higher abundance of Firmicutes and a stronger capacity to produce indoles and SCFAs than bacteria from PC compartment. Fiber supplementation significantly increased the production of SCFAs, indole-3-propionic acid, and indole-3-lactic acid, but decreased the production of oxindole, tryptamine, and serotonin. Pectin specifically promoted the production of indole-3-acetic acid and indole-3-aldehyde. Interestingly, supplementation of pectin or inulin increased the relative abundance of Bacteroidetes whereas supplementation of a mixture of two fibers decreased it. Overall, these results suggest that fiber supplementation and colon segment affect the composition of gut microbiota and the microbial catabolism of tryptophan.