Introduction: Gut pathogen colonization, where pathogens disrupt the normal gut microbiota, has been implicated in the development of bloodstream infections (BSIs). This study investigates the association between gut pathogen colonization and BSI, hypothesizing that species causing BSI primarily originated from gut. Methods: A prospective cohort study was conducted in the neonatal intensive care unit (NICU) of tertiary care hospital in Karnataka, India, from January 2021 to September 2023. Inborn preterm infants were enrolled. The study population was divided into two groups: group A (neonates without sepsis) and group B (neonates with sepsis). Demographic details and blood culture results were collected. Stool samples were taken on day 4 and day 14 for group A, and on day 4 and the day of sepsis diagnosis for group B. Results: Group B had a lower mean birthweight (1,649.6 ± 652.1 g) compared to group A (1,757 ± 656 g). Klebsiella pneumoniae was the most common pathogen causing BSIs (44.1%). The analysis revealed a high abundance of potential pathogens in the gut microbiome of group B neonates, with a concurrent decrease in beneficial gut flora. Conclusion: This study provides strong evidence for the association between gut pathogen colonization and BSI development in preterm neonates in NICUs. Gut microbiota modulation may serve as preventive strategy against BSIs, emphasizing the need for further research in this area to improve outcomes in vulnerable population.