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Pequi is a native and popular fruit in Cerrado biome. The internal yellow-orange mesocarp is the edible fraction of the fruit, but its shell (peel and external mesocarp), which comprises 80% of the fruit, is not used by the agro-industry during fruit processing. There is a growing interest in the reduction of food loss and waste because of environmental, economic, and social impacts. So this study evaluated the chemical composition, antioxidant capacity, and in vitro prebiotic activity of pequi shell flour. Pequi shell flour was obtained from the lyophilization and milling of pequi shell. The content of dietary fibers, oligosaccharides, sugars, organic acids, total phenolics and tannins, polyphenol profile, and antioxidant capacity was determined in pequi shell flour. In addition, its prebiotic activity was evaluated on growth and metabolism of probiotics Lactobacillus and Bifidobacterium strains. Pequi shell flour has a high content of dietary fibers (47.92 g/100 g), soluble fibers (18.65 g/100 g), raffinose (2.39 g/100 g), and phenolic compounds (14,062.40 mg gallic acid equivalents/100 g). For the first time, the polyphenols epigallocatechin gallate, epicatechin, and procyanidin B2 were identified in this by-product. Pequi shell flour promoted greater growth of Lacticaseibacillus casei L-26 (at 24–48 h) and Bifidobacterium animalis subsp. lactis BB-12, as well as higher prebiotic activity scores than fructooligosaccharides (standard prebiotic). Pequi shell flour is rich in prebiotic compounds and has a high antioxidant and prebiotic potential. The promising results encourage its use as an ingredient with antioxidant and potential prebiotic properties to elaborate new functional foods and nutraceuticals. [ABSTRACT FROM AUTHOR]