Background: As infection is a major cause of morbidity and mortality in neonates, early diagnosis and prompt treatment can prevent its serious consequences. The present study was conducted to determine the prevalence of infections in neonatal intensive care unit (NICU) of a tertiary care hospital and to study their risk factors, causative organisms and antimicrobial susceptibility pattern.Methods: Appropriate samples were collected from all neonates with clinical signs and symptoms of infections. Isolation of microorganisms, their identification and antimicrobial susceptibility was done according to standard microbiological techniques.Results: Among 1210 neonates admitted in the NICU, 393 (32.4%) were clinically suspected infections. The prevalence of Septicemia, Pneumonia, and Meningitis were 6%, 1.5%, 0.7% respectively. The predominant organisms causing neonatal infection were Gram negative bacteria followed by fungi and Gram positive bacteria. Among Gram negative bacteria, the antimicrobial resistance was highest for third generation Cephalosporins [Ceftazidime (81.1%), Cefotaxime (60.3%)]. In Gram positive bacteria highest resistance was observed for Penicillin and Ampicillin (91.3%). Methicillin resistance was observed in 91.6% of Coagulase negative Staphylococci (CoNS). All isolates of Candida parapsilosis were sensitive to Fluconazole, Voriconazole but resistant to Amphotericin B. Predominant risk factors were low birth weight (87.7%) and prematurity (75%). Maternal risk factors were pregnancy induced hypertension (13.4%) and premature rupture of membranes (PROM) (10.1%). The case fatality rate was 20.7%.Conclusions: There is a need of strict infection control measures and rational antibiotic policy to reduce the economic burden of hospital and community due to neonatal infections.