Introduction: Enteral feeding tubes are commonly utilized in the neonatal intensive care unit (NICU) due to the underdevelopment of coordination between sucking, swallowing, and breathing, or due to various diseases affecting the respiratory, cardiovascular, gastrointestinal, or neurological systems. The aim of the study was to investigate the differences in bacterial colonization patterns in hospitalized infants who received enteral feeding through either nasogastric (NG) or orogastric (OG) feeding tubes. Materials and Methods: This study was a hospital-based cross-sectional investigation that was carried out on infants who were admitted to the NICU. The NICU gathered feeding tubes throughout 4 months, beginning in August 2022 and continuing through December 2022. In addition, informed consent was collected from the participant’s parents or legal guardians for the entire group of people who took part in the research. The fact that the patient participated in this study had no bearing on the level of care that they were given. The research team gave serious consideration to recruiting any of the newborns who were receiving medical attention in the NICU and who had a feeding tube inserted during the time that sample collection was taking place. The infants did not receive any probiotics as a form of treatment in any of the cases. Results: Infants with NG tubes had a significantly higher number of C-sections ( P = 0.04), infants with NG tube were exclusively breastfed ( P = 0.02), and one or more dose of antenatal steroids was administered more in infants with OG tube ( P = 0.04). The most common types of bacteria found in NG and OG tubes were Proteobacteria (with a mean relative abundance of 50% and 60%, respectively), Firmicutes (with a mean relative abundance of 33% and 23%, respectively), and Actinobacteria (9% and 7%, respectively). Conclusion: Oral versus nasal feeding tube insertion was found to have a greater influence on bacterial colonization than any of the other clinical factors that were evaluated.