Several factors are known to influence the early colonization of the gut in newborns. Among them, the use of antibiotics on the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has scarcely been investigated, although this practice is routinely used in group B Streptococcus (GBS)-positive women. This work is therefore aimed at verifying whether IAP can influence the main microbial groups of the newborn gut microbiota at an early stage of microbial establishment. Fifty-two newborns were recruited: 26 born by mothers negative to GBS (control group) and 26 by mothers positive to GBS and subjected to IAP with ampicillin (IAP group). Selected microbial groups (Lactobacillus spp., Bidobacterium spp., Bacteroides fragilis, Clostridium difficile, and Escherichia coli) were quantified with real-time PCR on DNA extracted from newborn feces. Further analysis was performed within the Bidobacterium genus by using DGGE after amplification with genus-specific primers. Results obtained showed a significant decrease of the bifidobacteria counts after antibiotic treatment of the mother. Bifidobacteria were found to be affected by IAP not only quantitatively but also qualitatively. In fact, IAP determined a decrement in the frequency of Bidobacterium breve, Bidobacterium bifidum, and Bidobacterium dentium with respect to the control group. Moreover, this study has preliminarily evaluated that some bifidobacterial strains, previously selected for use in infants, have antibacterial properties against GBS and are therefore potential candidates for being applied as probiotics for the prevention of GBS infections.