Nasogastric feeding tubes (NG-tubes) from neonates contain potentially pathogenic bacteria. Using culture-based techniques, we have previously determined that the usage duration of NG-tubes did not impact the colonization of the nasogastric tubes. In the present study, we performed 16S rRNA gene amplicon sequencing to evaluate the microbial profile of 94 used nasogastric tubes collected from a single neonatal intensive care unit. Using culture-based whole genome sequencing, we as-sessed whether the same strain persisted in NG-tubes collected from the same neonate across different time-points. We found that the most commonly occurring Gram-negative bacteria wereEnterobacteriaceae,KlebsiellaandSerratia, while the most common Gram-positive bacteria were staphylococci and streptococci. The microbiota of the NG-feeding tube was overall infant-specific, rather than dependent on the duration of use. Furthermore, we determined that reoccurring species from the individual infant represented the same strain and that several strains were common for more than one infant. Our findings indicate that bacterial profiles found in NG-tubes of neonates are host-specific, not dependent on the duration of use and strongly influenced by the environment.