Premature infants in neonatal intensive care units (NICUs) are highly susceptible to infection due to the immaturity of their immune systems, and nosocomial infections are a significant risk factor for death and poor neurodevelopmental outcome in this population. To investigate the impact of cleaning within a NICU, a high-throughput short-amplicon-sequencing approach was used to profile bacterial and fungal surface communities before and after cleaning. Intensive cleaning of surfaces in contact with neonates decreased the total bacterial load and the percentage of Streptococcus species with similar trends for total fungal load and Staphylococcus species; this may have clinical relevance since staphylococci and streptococci are the most common causes of nosocomial NICU infections. Surfaces generally had low levels of other taxa containing species that commonly cause nosocomial infections (e.g., Enterobacteriaceae) that were not significantly altered with cleaning. Several opportunistic yeasts were detected in the NICU environment, demonstrating that these NICU surfaces represent a potential vector for spreading fungal pathogens. These results underline the importance of routine cleaning as a means of managing the microbial ecosystem of NICUs and of future opportunities to minimize exposures of vulnerable neonates to potential pathogens and to use amplicon-sequencing tools for microbial surveillance and hygienic testing in hospital environments. P remature infants in neonatal intensive care units (NICUs) are highly vulnerable to infections due to the immaturity of virtually every aspect of their innate and adaptive immune systems. The importance of hospital surfaces and the cleaning of these surfaces in interrupting outbreaks of specific organisms in NICUs (1, 2) and in eliminating reservoirs of potential pathogens (3-5) has been emphasized. However, most studies to date have relied on culture-based techniques, which can give incomplete and biased assessments of mixed microbial communities (6).The increasing ease and decreasing costs of high-throughput short-amplicon-sequencing technologies are propelling efforts to describe the microbial communities of surfaces on a massive scale (7). By parallel sequencing of short amplicons, typically 16S rRNA genes in bacteria and internal transcribed spacer (ITS) domains in fungi (8), it is possible to comprehensively profile the microbial composition of diverse environments. Microbial communities in intensive care units (ICUs) (9), hospitals (10), public restrooms (11), wineries (12), and office spaces (13) have been explored using high-throughput amplicon sequencing, advancing our knowledge of microbial trafficking and colonization in these systems. A recent report on bacterial communities on the surfaces in two NICUs demonstrated significant diversity and supported the hypothesis that colonies of pathogens on NICU surfaces may increase the risk of infection for premature infants (14).Stringent cleaning protocols for the surfaces in NICUs have been in place for many years, ...