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A retrospective study of bacterial isolates from cases of neonatal septicemia was undertaken over a period of 5 years (July 1998-June 2003) at the Government Medical College Hospital, Chandigarh, India. The study was carried out to determine the bacterial profile, the antimicrobial susceptibility of the isolates, and the change in trends over the study period. A total of 3,064 blood samples for blood culture were obtained, out of which 588 were positive for bacterial isolates. Most of the cases detected by blood culture occurred in the first week of life (64.4%). Gram-negative bacilli (58.5%) predominated over Gram-positive cocci (41.5%). Staphylococcus aureus was found to be the most common isolate (35.0%). The incidence of Gram-positive and Gram-negative organisms changed little over the 5 year span. However, a constant and significant rise in the incidence of Acinetobacter spp. was observed between the first to fifth year of the study period. Amikacin was found to be the most effective drug against Gram-negative bacteria. For S. aureus and Pseudomonas aeruginosa, overall resistance percentages of 5 years show that netilmicin and ciprofloxacin, respectively, were the most effective drugs.