The aims of this study were to determine the frequency and antimicrobial resistance patterns of microorganisms associated with sepsis cases in a public maternity hospital, and to analyze the main demographic data relating to these infected neonates over a two year period. We analyzed 255 positive blood cultures and the medical records of newborns admitted to the neonatal intensive care unit of the Hospital Maternidade Alexander Fleming II, Rio de Janeiro city, Brazil, from July 1997 to July 1999. Identification and antibiograms of the isolated strains were performed according to routine laboratory procedures. Demographics and microbiological data were analyzed using the Epi-Info program. The mean age of the newborns was 13.1 days, with an average of 1.2 strains isolated per patient. Antibiotics were administered to 207 (83.1%) patients before positive blood culture presentation. A total of 90.8% patients were premature; 83.9% had a gestational age of less than 36 weeks; 52.6% presented very low birth weight; 39.8% had asphyxia and 33.3% presented hyaline membrane disease. A total of 301 microorganisms were isolated with a predominance of Klebsiella pneumoniae (22.9%), coagulase-negative Staphylococci (17.3%), Serratia marcescens (15.9%) and Pseudomonas aeruginosa (10.6%). Gram-negative strains showed high resistance levels to cephalosporins and aminoglycosides. A total of 93.3% Staphylococcus aureus strains were methicillin-resistant. The newborns examined in this study presented several risk factors for acquiring hospital infection and the isolated microorganisms showed high levels of resistance to the majority of the antibiotics routinely used in the hospital.