OBJECTIVE: To determine the prevalence of nosocomial infection (NI) in newborns (NB) as well as the etiology, frequency mortality related to these infections in a tertiary-care hospital. MATERIAL AND METHODS: A retrospective epidemiological study was carried out with NB admitted to a tertiary-care neonatology hospital service from January 2006 to December 2008 who complied with selection criteria. All NB between the gestational ages of 25 to 44 weeks, NI supported by positive culture and either local or systemic infection were included. Descriptive statistic was used. RESULTS: NI prevalence in the Neonatal Service was an average of 30.4%. The most common microorganisms isolated in first event cultures (n = 100) were Gram-positive bacteria such as coagulase-negative staphylococci found in 55 patients (55%), followed by Gram-negative bacteria present in 44 patients (44%) and fungi such as Candida albicans and Candida sp. from 8 patients (8%); as to the second event (n = 32), Gram-negative bacteria were isolated from 20 patients (62.5%), Gram-positive bacteria such as coagulase-negative staphylococci were present in 6 patients (18.7%) and fungi such as Candida albicans were found in 5 patients (15.6%). Finally, regarding the third event (n = 18), Gram-negative bacteria were present in 16 patients (88.9%), Gram-positive bacteria were found in 15 patients (83.3%) such as coagulase-negative staphylococci in 10 patients (55.5%), and fungi such as Candida sp. in two (11.1%). Twelve (12%) who died secondary to IN, two suffered three NI events. CONCLUSIONS: Awareness of the various characteristics of NI in the work area (etiology, prevalence, and final outcome) is of great importance to design new interventions.