Original Research Article Objective: In this study our main goal is to evaluate bacteriological profile of neonatal sepsis in preterm babies of tertiary medical college and hospital. Method: this was a prospective observational and experimental study was carried out at Sir Salimullah Medical College and Mitford Hospital, Dhaka from July 2010 to October 2011 among 80 preterm babies. Results: The mean (±SD) weight of the babies was 1713.8 (±436.0) gm with a range of 500-2750 gm, 4(5.0%) babies were extreme preterm (gestational age <28 weeks), 12(15.0%) babies were very preterm (gestational age 28 to <32 weeks) and 64(80.0%) babies were preterm (gestational age 32 to <37 weeks). Ampicillin 71(88.8%)andgentamicin71(88.8%) were used in most of the cases, followed by ceftazidime35(43.8) and amikacin in 35(43.8%) cases, meropenem in 8(10.0%) cases, ciprofloxacin in 7(8.8%) cases, metronidazole in 11 (13.8%) cases. Among the organisms, Acinatobacter were present in 2 (2.5%) patient, Pseudomonas were present in 2(2.5%), CONS were present in 2(2.5%), Citrobacter in 1(1.3%) patient. Conclusion: From our study we can say that Acinatobacter, Pseudomonas and CONS were the most common isolate in our study. Early diagnosis and therapy are essential for the prevention of morbidity and mortality of neonatal sepsis in the neonatal intensive care unit.