Xylooligosaccharide (XOS) is an attractive prebiotic mainly due to its bifidogenic effect. However, commercial XOS with different compositions is often applied in the food industry at different doses without specifications. In this study, we evaluated the bifidogenic activity of XOS at different doses with either mixtures or pure fractions with different degrees of polymerization (DP), using three strains of spp., including ATCC 15700, ATCC 29521, and subsp. HN019. Three growth indicators showed strain-specific bifidogenic activity of XOS, and the activity was both dose- and fraction-dependent as only certain fractions stimulated significant growth. Adding 0.25% XOS (w/v) also promoted increase in total bifidobacterial population of rat fecal samples fermented . Albeit the antibacterial activity of XOS fractions can be demonstrated, significant growth inhibition can only be achieved when 4.0% XOS mixture was added in ATCC 6538 pure culture. In contrast, in the presence of HN019, 1.0% XOS showed significant antibacterial activity against ATCC 6538 in milk. In addition, RNA sequencing suggested downregulation of genes involved in ATCC 6538 infection, pathogenesis, and quorum sensing, by XOS. In conclusion, the report urges scientific specifications on XOS chemistry for its effective application as a novel food ingredient or functional food and provides novel insights into its bifidogenic and antibacterial activities.