Infants cared for in neonatal intensive care units (NICUs) are at a high risk for hospital acquired infections caused by bacteria and viruses. Despite the high risk of complications seen in these infants from infections, little is known about the bacterial diversity associated with NICU environments. In this study, we used culture-independent methods to assess the diversity of bacterial contamination on fomite surfaces in two NICUs. Our use of powerful culture-independent methods showed that NICU surfaces house many potential opportunistic pathogens and that these environments are dynamic niches for bacterial species. Our data also suggest areas within NICUs where contamination can be reduced by alternate cleaning practices.