There is growing awareness that interpersonal and age-dependent differences in gut microbiota composition impact prebiotic effects. The present study investigated the age-dependent prebiotic effect of fructans and HMOs in children and adults. For the fructans, inulin and FOS are well-established prebiotics. Human milk oligosaccharides (HMOs), on the other hand, are a more recent class of prebiotics that consist of a broader range of monosaccharide components and glycosidic linkage types, impacting their utilisation by the microbiota. Relying on the high throughput of the ex vivo SIFR® technology, all these actives were investigated in parallel on the gut microbiota of children and adults. The resulting compositional shift can be divided into three categories: bifidogenic across ages, for children, or for adults. Furthermore, age-specific compositional shifts occurred (B. pseudocatenulatum, and various Bacteroides spp for children; B. adolescentis, and various Phocaeicola spp). Lastly, by combining targeted analysis and untargeted metabolomics, health-promoting metabolites connected to the gut-brain axis, immunity, and overall gut health were found to be associated with those compositional changes. In conclusion, HMOs are promising modulators of the adult and particularly the children’s microbiota.