This study was designed to describe the epidemiology and risk factors for nosocomial infection (NI) in a Brazilian neonatal intensive care unit (NICU). This study was a retrospective cohort from January to December, 2003. All neonates admitted to the NICU. Infection surveillance was conducted according to the NNIS, CDC. Chi-square test and logistic regression model were performed for statistical analyses. The study was conducted at a public, tertiary referral NICU of a teaching hospital in the Northeast of Brazil. A total of 948 medical records were reviewed. Overall NI incidence rate was 34%. The main neonatal NI was bloodstream infection (68.1%), with clinical sepsis accounting for 47.2%, and pneumonia was the second most common NI (8.6%). Multivariate analysis identified seven independent risk factors for NIs: birth weight, exposure to parenteral nutrition, percutaneous catheter, central venous catheter or mechanical ventilation, abruptio placentae and mother's sexually transmitted disease (STD). Neonates from mothers with STD or abruptio placentae, those weighing less than 1,500 g at birth or those who used invasive devices were at increased risk for acquiring NI.