AbstractGalacto‐oligosaccharides (GOS) are prebiotic compounds, widely used as ingredients in various food, nutraceutical and pharmaceutical products. Enzymatic synthesis of GOS results in low‐purity products that contain high amounts of glucose and lactose beside the valuable GOS. In this study, a systematic approach was used to develop yeast‐based fermentation strategies to purify crude GOS. Potentially applicable yeast strains were identified based on an extensive search in literature databases followed by a series of laboratory‐scale fermentation tests. Single‐ and two‐step fermentation processes were designed for the removal of glucose alone or together with lactose from crude GOS syrup. Single‐step fermentation trials with two strains of previously unreported species, Cyberlindnera jadinii NCAIM Y.00499 and Kluyveromyces nonfermentans NCAIM Y.01443, resulted in purified products free of both glucose and ethanol from a crude GOS syrup diluted to 15 and 10 w/v%, respectively. Simultaneous removal of glucose and lactose was achieved by Kluyveromyces marxianus DMB Km‐RK in a single‐step fermentation process with a yield of 97.5% and final purity of 100%. A two‐step fermentation approach was designed to allow conversion of a glucose‐free product into a high‐purity GOS by removing glucose with C. jadinii Y.00499 in the first step, and lactose by Kluyveromyces lactis DMB Kl‐RK in the second step, resulting in a final product with a yield of 100% and a final purity of 92.1%. These results indicate that the selected nonconventional yeasts are promising candidates for the removal of non‐GOS components from commercial crude GOS products by selective fermentation.