The prebiotic effects of hyaluronan (HA) are widely recognized, contributing to improved gut health and immune modulation. Despite its extensive use as dietary supplement, the specific interactions between HA oligosaccharides (o-HAs) and the gut microbiome remain largely unexplored. To investigate its role and metabolic fate in gut homeostasis, 200 mg/day of o-HAs (average molecule weight 1 kDa) were added to an automated computer-controlled SIMulator of the Gastrointestinal tract (SIMGI). The results revealed a significant reshaping of the intestinal flora composition by o-HAs, notably reducing the Firmicutes/Bacteroides ratio. Fermentation of o-HAs by gut microbiota significantly increased the abundance of Bifidobacterium, Prevotellaceae_Prevotella, Dialister, Eubacterium, and Sutterella, but decreased that of Catenibacterium, Oscillospira, Klebsiella, and Citrobacter (P < 0.05). This corresponded with significant enhancements in the content of short-chain fatty acids (SCFAs) such as acetic acid, propionic acid and n-butyric acid, highlighting the significant impact of o-HAs at the genus level. Furthermore, analysis of microbial function predicted the downregulation of pathological events in nine human diseases, particularly infectious ones (parasitic and bacterial). Potential inhibitions were observed in metabolic pathways associated with pentose and glucuronate interconversions as well as cationic antimicrobial peptide resistance. These findings underscore the in vitro prebiotic effects of o-HAs and their potential relevance in managing diverticular diseases or preventing metabolic disorders through the regulation of gut microbiota.