Introduction: A condition in which bacteria, viruses or fungi causes hemodynamic changes in the neonates and results in morbidity and mortality is called Neonatal sepsis. The symptoms of neonatal sepsis are nonspecific but some of them are respiratory distress convulsions and temperature dysregulation. Aim: To determine the Neonatal Sepsis and its Bacteriological Profile in a tertiary care hospital: Methodology: All neonates who were suspected of having neonatal sepsis were included in the study. Sepsis was diagnosed through clinical examination. Some clinical features were observed such as fever, lethargy, respiratory distress, jaundice and umbilical infections. Under aseptic conditions, sepsis cultures and screens were sent. Analyzation of data was done using STATA version 11. Chi-square test was used to analyze clinical features. Descriptive statistics was helpful in analyzing bacteriological profile. Results: A total of 2325 neonates were admitted to the hospital. Out of these admissions, 312 were eligible for clinical sepsis. The blood culture positivity rate was 12%. Early-onset sepsis was present in 52.2% cases. Culture positive rate in late-onset sepsis was increased due to low birth weight, prematurity and parenteral nutrition. Positive screen had sensitivity of 80.1% and negative value of 85.6%. Isolation of gram-negative organisms was more common. Klebsiella pneumoniae and Acinetobacter were the commonly isolates. Conclusion: Neonatal sepsis was associated with low birth weight, low APGAR score, preterm and parenteral nutrition. Klebsiella pneumoniae, Acinetobacter and Staphylococci are the main causative organisms. Gram-negative bacteria were resistant to a wide range of medications.