Objective: Identification of pathogens associated with early onset neonatal sepsis and their resistance pattern so that a guideline for empirical antibiotic regimen can be formulated. Methods: Study was conducted in a teaching hospital in eastern part of Bengal from March 2011 to August 2012. Newborn, age less than 72hours, associated with a clinical diagnosis of sepsis were enrolled in this study. 1 ml of blood was collected maintaining proper asepsis and inoculated in 5 ml of brain heart infusion broth. The broth was incubated aerobically at 370 C and was sub cultured in blood agar, chocolate agar and MacConkey__ampersandsignrsquo;s agar. Isolates were identified by colonial morphology, Gram staining and standard biochemical tests. Antimicrobial sensitivity test was performed by Kirby Bauer disc diffusion method following CLSI guidelines. Result: Among 192 clinically suspected cases of early onset neonatal sepsis, blood culture was positive in 73(38.0%) cases of which Klebsiella pneumoniae (45.71%) was the predominant organism followed by Staphylococcus aureus (20%), Escherichia coli (11.42%), Enterococcus sp (7.14%), Enterobacter sp (5.71%) etc. Most Gram negative organisms were highly resistant to ampicillin (100%), gentamicin (81.25%) and cephalosporin group of drugs. Imipenem (0%) and ofloxacin (31.25%) were the two most effective drugs against these gram negative bacteria. 50% of Staphylococcus aureus were methicillin resistant. Conclusion: This study reflected alarmingly increasing in vitro resistance of isolated organisms towards commonly used antibiotics specially ampicillin, gentamicin and third generation cephalosporins which were given empirically in neonatal sepsis. Therefore this study recommends introduction of fluoroquinolone along with amikacin in empirical treatment of early onset neonatal sepsis.