Candida lusitaniae is an increasingly important nosocomial bloodstream pathogen. Epidemiological investigation and molecular typing techniques identified three neonates infected with identical strains of C lusitaniae that were distinguished readily from epidemiologically unrelated strains from other locations in the hospital. The results of this study provide evidence for nosocomial transmission of C lusitaniae in a neonatal intensive-care unit and suggest that these infants are at increased risk for infection with this agent.