OBJECTIVE: To determine the most important causative agents of bacteremia in a neonatal intensive care unit (NICU); their changing distribution and their antibiotic susceptibility patterns over a 5-year period. METHODS: This study was performed between January 1997 and January 2001 at the Microbiology Section, Tripoli Medical Center, Tripoli, Libya. During this period 1431 Oxoid Signal Blood Cultures sets were obtained from 1092 NICU with suspected bacteremia. Conventional methods, API 20 E and API 20 NE were used to identify the isolated bacteria. The Kirby-Bauer disk diffusion method was performed to assess their antibiotic susceptibilities in accordance with National Committee for Clinical Laboratory Standards. RESULTS: During the study period, 801 sets out of the total 1431 blood cultures were positive for microbial growth, which represented 648 cases of neonatal bacteremia from a total of 1092 cases. From the total number of isolates, the members of Enterobacteriaceae Serratia, Klebsiella and Enterobacter spp were the most common cause of bacteremia. The coagulase negative and positive Staphylococci were also frequently isolated. A changing pattern of causative pathogen was observed during this study between members of the 3 leading genera of Enterobacteriaceae. Antibiotic susceptibility testing showed a high level of resistance among the most common pathogens. Resistance to aztreonam, imipenem, ciprofloxacin and piperacillin/tazobactam was less frequently encountered. Staphylococcus resistance to anti-staphylococcal antibiotic and due to hyperproduction of penicillinase enzyme was also high and all isolates were remained sensitive to vancomycin. CONCLUSION: Gram-negative bacteria especially members of the Enterobacteriaceae are important causes of bacteremia in NICUs. Although most isolates remain sensitive to the new antibiotics, emergence of resistant strains cannot be excluded in the future. For that reason, new strategies and continuous surveillance are required to monitor the changing epidemiology of pathogens, antibiotic susceptibilities and antibiotic use needed to overcome the increasing incidence of resistance to conventional drugs.