Cashew apple by-product (CAB) is an important agro-industrial waste still underutilized, although it has been characterized as source of a variety of nutrients and bioactive compounds. This study evaluated the capability of freeze-dried CAB (FCAB) submitted to a simulated gastrointestinal digestion of inducing changes in relative abundance of distinct microbial groups found as part of human colonic microbiota, as well as in pH and short-chain fatty acid production during a 24-h in vitro fermentation using a pooled human fecal inocula. FCAB increased the relative abundance of Bifidobacterium and Lactobacillus/Enterococcus during colonic fermentation, besides to decrease the relative abundance of Bacteroides/Prevotella, Eubacterium rectale/Clostridium coccoides, and Clostridium histolyticum. FCAB increased the counts of lactic acid bacteria and decreased the counts of Enterobacteriaceae during colonic fermentation. Furthermore, FCAB decreased pH and increased the production of short-chain fatty acids in colonic fermentation media. These effects could be linked to contents of dietary fibers and the presence of fructans and different phenolic compounds found in FCAB. These results showed that FCAB induced positive alterations in composition and metabolic activity of human colonic microbiota in vitro, which indicate prebiotic properties.