Neonatal necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among very low birth weight infants. It has long been suspected that intestinal microbes contribute to the pathogenesis of NEC, but the details of this relationship remain poorly understood. Recent advances in molecular biology and enteric microbiology have improved our ability to characterize intestinal microbes from infants with NEC and from healthy unaffected newborns. The lack of diversity within the neonatal intestine makes it possible to study gut microbial communities at a high level of resolution not currently possible in corresponding studies of the adult intestinal tract. Here, we summarize clinical and laboratory evidence that supports the hypothesis that NEC is a microbe-mediated disorder. In addition, we detail recent technologic advances that may be harnessed to perform high-throughput, comprehensive studies of the gut microbes of very low birth weight infants. Methods for characterizing microbial genotype are discussed, as are methods of identifying patterns of gene expression, protein expression, and metabolite production. Application of these technologies to biological samples from affected and unaffected newborns may lead to advances in the care of infants who are at risk for the unabated problem of NEC.