Recently, prebiotic supplementation of infant formula has become common practice; however the impact on the intestinal microbiota has not been completely elucidated. In this study, neonatal piglets were randomized to: formula (FORM, n = 8), formula supplemented with 2 g/L each galactooligosaccharides (GOS) and polydextrose (PDX, F+GP, n = 9) or a sow-reared (SOW, n = 12) reference group for 19 days. The ileal (IL) and ascending colon (AC) microbiota were characterized using culture-dependent and -independent methods. 16S amplicon sequencing identified no differences at the genera level in the IL. Interestingly, six genera in the AC were significantly different between FORM and F+GP (P<0.05): Lactobacillus, Ruminococcus, Parabacteroides, Oscillospira, Hydrogenoanaerobacterium and Catabacter. In particular, the relative abundance of AC Lactobacillus was higher (P = 0.04) in F+GP as compared to FORM. Culture-dependent analysis of the IL and AC lactobacilli communities of FORM and F+GP revealed a Lactobacillus spp. composition similar to 16S amplicon sequencing. Additional analysis demonstrated individual Lactobacillus isolates were unable to ferment PDX. Conversely, a majority of lactobacilli isolates could ferment GOS, regardless of piglet diet. In addition, the ability of lactobacilli isolates to ferment the longer chain GOS fragments (DP 3 or greater), which are expected to be present in the distal intestine, was not different between FORM and F+GP. In conclusion, prebiotic supplementation of formula impacted the AC microbiota; however, direct utilization of GOS or PDX does not lead to an increase in Lactobacillus spp.