Coagulase-negative staphylococci, especially Staphylococcus epidermidis, are increasingly important nosocomial pathogens, particularly in critically ill neonates. A 3-year prospective surveillance of nosocomial infections in a neonatal intensive care unit (NICU) was performed by traditional epidemiologic methods as well as molecular typing of microorganisms. The aims of the study were (i) to quantify the impact of S. epidermidis on NICU-acquired infections, (ii) to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species, (iii) to evaluate the use of different methods for the epidemiologic typing of the isolates, and (iv) to characterize the occurrence and the spread of staphylococci with decreased glycopeptide susceptibility. Results confirmed that S. epidermidis is one of the leading causes of NICU-acquired infections and that the reduced glycopeptide susceptibility, if investigated by appropriate detection methods such as population analysis, is more common than is currently realized. Typing of isolates, which can be performed effectively through molecular techniques such as pulsed-field gel electrophoresis but not through antibiograms, showed that many of these infections are due to clonal dissemination and, thus, are potentially preventable by strict adherence to recommended infection control practices and the implementation of programs aimed toward the reduction of the unnecessary use of antibiotics. These strategies are also likely to have a significant impact on the frequency of the reduced susceptibility of staphylococci to glycopeptides, since this phenomenon appears to be determined either by more resistant clones transmitted from patient to patient or, to a lesser extent, by strains that become more resistant as a result of antibiotic pressure.