Background: This study explores the potential of food industry by-products, such as plant peels, stems, and slags, as valuable sources of lignocellulosic material (LCM), which contains 25-40% xylan. These underutilized resources, often discarded as waste, hold the promise of sustainable applications in biotechnology. By safely extracting xylooligosaccharides (XOS) from LCM biomass, the value of these materials can be significantly enhanced, contributing to green production and supporting sustainable development. XOS, recognized for its prebiotic activity, has been shown to promote the growth of beneficial gut bacteria, making it a vital research area in the fields of food science, medicine, and technology. Aim: To extract and characterize oligosaccharides derived from by-products of the food industry, evaluate their physicochemical properties, and investigate selected biological activities. Materials and Methods: This study utilized microwave pretreatment and enzymatic hydrolysis to isolate and purify XOS from wheat bran and brewers’ spent grains (BSG), provided by Altan Taria LLC and APU CoL, respectively. Microwave irradiation at 200°C for 5 minutes was employed as a pretreatment step, followed by hydrolysis using commercial xylanase (Thermomyces lanuginosus, recombinant Aspergillus oryzae, 2500 BXU/g) at 55°C for 24 hours. The resulting hydrolysate underwent filtration with activated carbon and ethanol precipitation to yield purified XOS. Analytical methods, including FTIR spectroscopy, TLC and HPLC, were used for structural and compositional analysis of the purified oligosaccharides. In vitro tests evaluated the ability of XOS to support the growth of beneficial gut bacteria, including Bifidobacterium spp., Lactobacillus fermentum (ATCC 9338), and Lactobacillus casei (ATCC 344), using XOS-enriched media. Additionally, in vivo studies were conducted on rats to determine the biological effects of XOS on gut microbiota. Results: The results demonstrated that prolonged enzymatic hydrolysis for more than 10 hours, using 0.25 g of xylanase per 100 g of substrate, resulted in optimal yields. XOS purity was measured at 87.6% with an 8.1 g yield from wheat bran and 89% purity with a 7.2 g yield from brewers’ spent grains. Structural analysis confirmed the presence of xylobiose, xylotriose, and xylotetraose, with xylotetraose being the most abundant component in WBP-XOS (47.5%), and xylobiose dominating BGS’s derived XOS (47.8%). Biological effects revealed that wheat bran-derived XOS significantly supported the growth of Bifidobacterium spp. and L. fermentum (ATCC 9338) in a concentration-dependent manner, whereas no significant effect was observed on L. casei (ATCC 344). In vivo studies confirmed that XOS consumption increased populations of Bifidobacterium spp. and Akkermansia muciniphila spp. in gut microbiota (p<0.05). Furthermore, XOS consumption reduced plasma cholesterol, triglycerides, and LDL-C levels while increasing HDL-C levels, demonstrating metabolic benefits. Conclusion This research establishes that XOS with prebiotic activity can be efficiently extracted and purified from food industry by-products using microwave-assisted enzymatic hydrolysis. This approach highlights the potential of utilizing agricultural and industrial waste for producing functional prebiotics, contributing to sustainable practices and offering valuable applications in health and nutrition.