From November 2011 through March 2012, we surveyed 272 babies in our neonatal intensive care unit for rectal colonization with vancomycin-resistant enterococci (VRE). Using Spectra VRE medium (Remel Diagnostics, Lenexa, KS), we identified one neonate colonized with vancomycin-resistant Enterococcus faecium. In addition, 55 (13%) of the surveillance cultures yielded false-positive results with vancomycin-susceptible Enterococcus faecalis. During the same time period, 580 rectal swabs were collected from adult patients resulting in 20 (3%) false-positive cultures. The difference in false-positive rates between cultures from babies and adults was statistically significant (P < 0.001), prompting an investigation of factors that might influence the elevated false-positive rate in the neonates including patient demographics, nutrition, and topical ointments applied at the time of testing. Older neonates, with a median age of 6 weeks, were more likely to have false-positive cultures than younger neonates with a median age of 3 weeks (P < 0.001). The younger neonates receiving Similac Expert Care products were less likely to have false-positive surveillance cultures than those receiving other formulas (P < 0.001). Application of topical products was not associated with false-positive cultures. The false-positive E. faecalis strains were typed by Diversilab Rep-PCR (bioMérieux, Marcy l'Etoile, France) and found to represent eight different groups of isolates. The utility of the Spectra VRE media appeared to be significantly impacted by the age of the patients screened.