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This study aimed to investigate the specific growth stimulation of certain desired intestinal bacteria by a novel galacto-oligosaccharide mixture, which was produced with a &#946;-galactosidase from a potential probiotic <i>Lactobacillus</i> isolate that contained mainly oligosaccharides of &#946;-1,3 and &#946;-1,6 glycosidic linkages (termed Lb-GOS) using single-strain fermentations. The composition of this Lb-GOS mixture was 33.5% disaccharides, 60.5% trisaccharides, 4.8% tetrasaccharides, and 1.0% pentasaccharides with a negligible amount of monosaccharides, lactose, and lactobionic acid (0.3%). Eight <i>Lactobacillus</i> spp. strains and three <i>Bifidobacterium</i> spp. strains were used in single-strain fermentations to determine the fermentation activity scores of this Lb-GOS preparation compared to two commercially available prebiotic mixtures, 4&#8242;GOS-P and Vivinal GOS (V-GOS). The highest scores were obtained when <i>L. reuteri</i> Lb46 and the two <i>Bifidobacterium</i> strains, <i>B. animalis</i> subsp. <i>lactis</i> Bif1 and Bif3, were grown on these galacto-oligosaccharide mixtures. In addition, the Lb-GOS mixture was found to have higher fermentation activity scores; hence, it stimulated the growth of these probiotic strains more than 4&#8242;GOS-P and V-GOS, which may be attributed to the different glycosidic linkage types that are found in the Lb-GOS mixture compared to the other two commercial preparations. These findings suggested that the Lb-GOS mixture that is described in this work should be of interest for the formulations of new carbohydrate-based functional food ingredients.