The current screening study aimed at identifying promising prebiotic and synbiotic candidates. The fermentation of xylo-oligosaccharides, xylan, galacto-oligosaccharide, fructo-oligosaccharide, polydextrose, lactitol, gentiobiose and pullulan was investigated in vitro. The ability of these established and potential prebiotic candidates to function as a sole carbon source for probiotic (Bifidobacterium and Lactobacillus), intestinal and potential pathogenic microbes (Eubacterium, Bacteroides, Clostridium, Escherichia coli, Salmonella, and Staphylococcus) was assessed in pure cultures. Xylo-oligosaccharides were fermented with high specificity by the tested Bifidobacterium lactis strains and lactitol by lactobacilli, whereas galacto-oligosaccharides, fructo-oligosaccharides and gentiobiose were utilised by a larger group of microbes. Xylan, polydextrose and pullulan were utilised to a limited extent by only a few of the tested microbes. The results of this screening study indicate that xylo-oligosaccharides and lactitol support the growth of a limited number of beneficial microbes in pure cultures. Such a high degree of specificity has not been previously reported for established prebiotics. Based on these results, the most promising prebiotics and synbiotic combinations can be selected for further testing.