A study was performed to assess the incidence density of, and to identify the risk factors associated with, nosocomial bloodstream infection (BSI) in a neonatal intensive care unit (NICU) in Athens. Twenty-four of 105 patients developed nosocomial BSI (22.9%). The incidence density of BSI was 10.9 per 1000 patient-days. A multivariate model showed that only two factors were significantly and independently responsible for nosocomial BSI: central venous catheter use and umbilical catheter use. Results showed that the incidence density rate was high and the factors that had most influence on the development of nosocomial BSI were associated with the treatment received by neonates during their stay in the NICU. Therefore, surveillance of nosocomial BSI and strategies such as infection control, nursery design and staffing should be implemented to reduce the incidence of these infections. This effort should be multidisciplinary, involving staff who insert and maintain intravascular catheters, and healthcare managers who allocate resources.