This study investigated the effects of different delivery modes on oral microflora in healthy newborns immediately post-partum, and provided evidence for microbial colonization disruption induced by medical procedures. Eighteen infants delivered by cesarean section and 74 by vaginal delivery were included in the study. High-throughput sequencing of 16S bacterial rRNA was performed on oral samples collected immediately after birth. All data were analyzed using bioinformatics approaches. Our results indicated that different oral bacteria were found between infants delivered by cesarean section compared to vaginal delivery group. Lactobacillus, Prevotella and Gardnerella were the most abundant genera in the vaginal group, while Petrimonas, Bacteroides, Desulfovibrio, Pseudomonas, Staphylococcus, Tepidmicrobium, VadinCA02, and Bifidobacterium were dominant bacteria in the cesarean section (C-section) group. Furthermore, bacteria isolated from 27 vaginally-delivered infants were not clustered into the vaginal group. Most of them spent more than 24 hours in the delivery room and this led to repeated sterilization procedures. We hypothesized that repeated sterilization might have influenced oral microflora in those cases. To conclude, this study suggested that different modes of birth delivery affect oral microflora in healthy infants. In addition, attention shall be paid to the clinical practice of repeated sterilization of the vulva that possibly obstructs the colonization of vaginal bacterial.