Objective The objective of the study was to investigate the differences in the gut microbiomes of neonates delivered via cesarean section compared to those born by vaginal delivery, and to identify the predominant microbial taxa present in each group. Study design A prospective observational study. Setting At Her Royal Highness Maha Chakri Sirindhorn Medical Center, Srinakharinwirot University, Thailand. Sample Fecal sample obtained by 281 healthy neonates born between February 2021 and April 2023. The study population was divided into two groups: 139 neonates born via vaginal delivery and 142 neonates born via cesarean section. Methods The microbiota composition of each neonate’s fecal sample was identified by using 16S ribosomal ribonucleic acid metagenomic sequencing. Main Outcome Measures Neonatal gut microbiome abundancy and diversity was identified according to route of delivery. Results Neonates delivered vaginally exhibited a gut microbiome with higher abundance and diversity than those delivered by cesarean delivery. Bifidobacterium was the dominant genus in both groups. Bifidobacterium breve was the dominant species and was significantly higher in cesarean-delivered neonates compared to those delivered vaginally (24.0% and 9.2%, respectively) (p<0.0001). However, the taxonomy of only 89 (64.0%) and 44 (31.43%) fecal samples could be identified from the vaginal and cesarean delivery groups, respectively. Conclusions Route of delivery is associated with neonatal gut microbiome abundancy and diversity. Neonates delivered via vaginal delivery exhibited higher diversity but lower abundancy of the dominant species in the gut microbiome.