Raw transgalactosylated oligosaccharides (raw TOS) obtained by enzymatic synthesis with Aspergillus oryzae β-galactosidase were purified by selective bioconversion with Saccharomyces cerevisiae ATCC4126 and Kluyveromyces marxianus NRLLY-1109. Monosaccharides were completely removed by S. cerevisiae and K. marxianus from raw TOS without any nutrient supplementation. The effect of the biomass to carbohydrate mass ratio on the purity and specific productivity of removal of sugars was assessed with S. cerevisiae, where both effects were significant. Purity obtained with a collection strain of S. cerevisiae and bakers’ yeast was compared, the latter being higher after 24 h of bioconversion. A product free of monosaccharides was obtained with a purity (0.48; 28% lactulose and 20% oligosaccharides), similar to that of commercial galacto-oligosaccharide preparations. The possibility of yeast recovery and reuse in sequential batch operation was assessed: two batches could be performed before a significant reduction in monosaccharide removal was seen.