In this study, we conducted in vitro fermentation with infant faecal inocula dominated by Bifidobacterium longum, Bifidobacterium breve, and Bacteroides enterotypes, and assessed the efficacy of the metabolome associated with 2′-fucosyllactose (2′-FL) fermentation on lipopolysaccharide-induced epithelial cell barrier damage on Caco-2 cells. The results revealed that propionate and butyrate were more prominent in the Bacteroides-dominated group, whereas the Bifidobacterium-dominated group was associated with an increased molar fraction of lactate (∼16 mm). Additionally, our study demonstrated that 2′-FL faecal metabolites (FMs) decreased the production of inflammatory cytokines, and promoted tight junction gene expression by inhibiting the TLR4/NF-κB/MLCK signalling pathway and activating the Nrf2/HO-1 pathway. The 2′-FL FMs from Bacteroides-dominated enterotypes had a unique advantage in regulating the TLR4/NF-κB/MLCK signalling pathway, which can be attributed to the effects of propionate and butyrate.