Background: According to the research by the World Health Organization in 2021, the use of cesarean sections continues to rise globally, now accounting for more than 21% of all deliveries. Intrapartum antibiotic prophylaxis (IAP) is commonly used in cesarean delivery as well as in Group B Streptococcus-positive women (35%) prior to vaginal delivery. This study aimed to investigate the effect of the mode of delivery and exposure to antibiotics during delivery on neonatal gut bacteriome. Methods: Newborns (n = 66) from the CELSPAC: TNG cohort, who were born after the 38th week of pregnancy, were included in this study. Their fecal samples were collected within the first four days after the cesarean delivery with IAP (n = 18), vaginal delivery with IAP (n = 15), or without IAP (n = 33, the control group). The genomic DNA extraction with DNeasy® PowerSoil® kit was followed by creation of 16S rRNA gene library and sequencing performed using the MiSeq reagent kit V3 on a MiSeq 2000 instrument according to the manufacturer’s instructions. Results: When fecal samples were stratified according to the antibiotic exposure during delivery, a statistically significant difference in the number of observed species (p = 0.0035) and a trend in the bacterial diversity (Shannon index, p = 0.056) were found. However, there was no difference in these parameters depending on mode of delivery (p > 0.05). PERMANOVA test assessing the meconium and transitional stool separately revealed significant differences in neonatal transitional stool bacteriome (n = 31) among three studied groups according to the mode of delivery and antibiotic exposure during delivery (q = 0.001), but no significant differences in meconium samples (n = 35). Vaginal delivery without IAP was significantly associated with increased relative abundances of Bifidobacteria (q = 0.007), Bacteroides (q = 0.007), and decreased relative abundances of Rothia (q = 0.01) and Enterococcus species (q = 0.009) in transitional stool samples in comparison to cesarean-born and vaginally-born newborns with IAP. The results also indicate that the relative abundance of Rothia and Enterococcus species in transitional stool samples is affected by the mode of delivery rather than antibiotic exposure during delivery. Conclusion: The results indicate that the mode of delivery as well as IAP has profound effects on gut bacterial composition in the first days of newborns’ life.