Prebiotics are used for stimulating the growth of beneficial microorganisms in the gut. However, it is very difficult to find a suitable prebiotic mixture that exclusively supports the growth of beneficial microbes such as bifidobacteria and lactobacilli. We tested the effects of a prebiotic mixture in vitro by incubating it with fecal samples and in vivo by administration of the prebiotic supplement to healthy adult volunteers, followed by analysis of their fecal microbiota. The effect of the oligosaccharides on bacterial metabolism was studied by analyzing short-chain fatty acid (SCFA) production in vitro and the SCFA pattern for the stool samples of volunteers. In the in vitro test, a higher proportion of bifidobacteria (25.77%) was seen in the total bacterial population after cultivation on a prebiotic mixture than on the control medium (7.94%). The gram-negative anaerobe count significantly decreased from 8.70 to 6.40 log CFU/g (from 35.21% to 0.60%) and the Escherichia coli count decreased from 7.41 to 6.27 log CFU/g (from 1.78% to 0.44%). Administration of a prebiotic mixture in vivo (9 g of galactooligosaccharides [GOS] + 1 g of maltodextrins; daily for 5 days) significantly increased the fecal bifidobacterial count from 9.45 to 9.83 log CFU/g (from 40.80% to 53.85% of total bacteria) and reduced the E. coli count from 7.23 to 6.28 log CFU/g (from 55.35% to 45.06% of total bacteria). The mixture comprising GOS and maltodextrins thus exhibited bifidogenic properties, promoting the performance of bifidobacteria by boosting their growth and inhibiting the growth of undesirable bacteria.