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Infants born before 28 weeks are at risk of contracting healthcare-associated infections (HAIs), which could be caused by pathogens residing on contaminated hospital surfaces. In this longitudinal study, we characterized by NGS the bacterial composition of nasal swabs of preterm newborns, at the time of birth and after admission to the Neonatal Intensive Care Unit (NICU), comparing it with that of the environmental wards at the time of delivery and during the hospitalization. We characterized the resistome on the samples too. The results showed that environmental microorganisms responsible for HAIs, in particular Staphylococcus spp., Streptococcus spp., Escherichia-Shigella spp., and K. pneumoniae , were detected in higher percentages in the noses of the babies after 13 days of hospitalization, in terms of the number of colonized patients, microorganism amount, and relative abundance. The analysis of nasal bacteria resistome evidenced the absence of resistance genes at the time of birth, some of which appeared and increased after the admission in the NICU. These data suggest that hospital surface microbiota might be transported to respiratory mucosae or other profound tissues. Our study highlights the importance of a screening that allows characterizing the microbial profile of the environment to assess the risk of colonization of the newborn.