Objective: To analyse the Gram-negative bacteriological profile of nosocomial sepsis and antibiotic susceptibility patterns. Methods: Neonates clinically diagnosed with sepsis and whose blood cultures tested positive for Gram-negative microorganisms from 2002 to 2008, retrospectively. Results: 143 cases of neonatal sepsis caused by Gram-negative microorganisms were identified. Eighty-one percent (n=116) of these had nosocomial sepsis. In nosocomial sepsis, the most common isolated Gram-negative microorganism was Serratia marcescens (16.4%). Levofloxacin (97.4%), meropenem (97.1%), imipenem (95.6%), ciprofloxacin (95.4%) and amikacin (94.8) were the most sensitive antibiotics to Gram-negative micro-organisms. Sepsis-related mortality rate was 16% (n=23) in nosocomial sepsis caused by Gram-negative microorganisms. Conclusions: Carbapenem seems to be the best option for nosocomial sepsis caused by Gram-negative micro-organisms in our neonatal intensive care unit. Every unit must evaluate causative agents and antimicrobial susceptibilities in order to select the appropriate empirical therapy for nosocomial sepsis.