Xylooligosaccharides (XOS) are prebiotics of significant biological value that can be obtained through cost‐effective purification of agricultural waste. The present research featured in vitro and in vivo investigation of prebiotic effects of xylooligosaccharides derived from wheat bran powder and brewer's spent grain. Prebiotic activity of Lactobacillus. fermentum, Lactobacillus. casei, and Bifidobacterium spp. was investigated in vitro using standard selective media. 16S rRNA quantitative PCR used for in vitro and in vivo investigation quantified relative abundance of Bifidobacterium spp., Lactobacillus spp., and Akkermansia. muciniphila in samples of fecal matter, cecal content, and intestinal tissue. Research revealed a favorable association between XOS concentration and both bacterial count and diameter of resultant colonies. The standard strain of L. casei showed no noticeable effect on growth rate. Bifidobacterium spp. proliferation in intestinal tissue was validated via in vivo tests using XOS obtained from wheat bran powder and brewer's spent grain. Findings indicated increased prevalence of the A. muciniphila species and the presence of XOS showed a protective function in preserving the structural integrity of intestinal mucus secretions. The presence of XOS in food indicated direct association with proliferation of Bifidobacterium spp. and A. muciniphila spp. Study results suggest that XOS extracted through enzymatic hydrolysis in Mongolian food industry by‐products such as wheat bran products and brewer's spent grain exhibit prebiotic properties that justify XOS manufacture on a large scale and incorporation of XOS as nutritional enhancement in food products and pharmaceuticals.