Bacillus subtilis natto is a potential source of fructooligosaccharides (FOS), which can be obtained by fermentation and may stimulate the growth of beneficial microorganisms in the colon representing a strategy to manipulate the intestinal microbiota acting as a prebiotic compound. The present study focuses on the ability of Lactobacillus ssp. strains to utilize FOS as a sole energy source. The results showed that FOS was equally good as glucose to provide energy source. The highest prebiotic activity score was obtained with Lactobacillus plantarum ATCC 14917 grown on FOS (0.526), followed by Lactobacillus casei (LC-1) (0.222). The lowest score was for Lactobacillus paracasei ATCC 27092 (−0.051). The results suggests that specific combinations of probiotic (L. plantarum ATCC 14917 and L. casei (LC-1)) and prebiotic (FOS) could be used as synbiotics in dairy and other foods.