Enterococcus faecalis is a ubiquitous intestinal symbiont and common early colonizer of the neonatal gut. Although colonization with E. faecalis has been previously associated with decreased pathology of necrotizing enterocolitis (NEC), these bacteria have been also implicated as opportunistic pathogens. Here we characterized 21 strains of E. faecalis, naturally occurring in 4-day-old rats, for potentially pathogenic properties and ability to colonize the neonatal gut. The strains differed in hemolysis, gelatin liquefaction, antibiotic resistance, biofilm formation, and ability to activate the pro-inflammatory transcription factor NF-κB in cultured enterocytes. Only 3 strains, BB70, 224, and BB24 appreciably colonized the neonatal intestine on day 4 after artificial introduction with the first feeding. The best colonizer, strain BB70, effectively displaced E. faecalis of maternal origin. Whereas BB70 and BB24 significantly increased NEC pathology, strain 224 significantly protected from NEC. Our results show that different strains of E. faecalis may be pathogenic or protective in experimental NEC.