The present study evaluated the impact of high-intensity ultrasound processing of potentially prebiotic açai juice containing complex carbohydrates (dextran and oligosaccharides) on human gut microbiota after in vitro colonic fermentation. The juice was subjected to an in vitro simulated digestion and human fecal fermentation. HPLC quantified the metabolites produced during fecal fermentation, and the 16 S rRNA sequencing evaluated the microbiota change. The gluco-oligosaccharides and dextran were resistant to in vitro digestion and promoted the fecal microbiota production of short-chain fatty acids (SCFAs). The sonicated juice markedly increased propionic and butyric acid production, reaching 11 g/L and 4 g/L, respectively. The potentially prebiotic açai juice improved the richness (25%) and diversity (22%) of the fecal microbiota, and the ultrasound processing increased richness (12%) compared to non-sonicated juice, which may contribute to overall host health. The study demonstrated that açai juice containing gluco-oligosaccharides and dextran has a potential prebiotic effect. Incorporating this juice into the diet might promote gut-beneficial bacterial growth, improving the gut microbiota and the health-promoting metabolites.