Background: Neonatal sepsis is one among the leading cause for neonatal mortality rate and morbidity in neonatal ward and neonatal medical care unit especially in developing countries. Frequent monitoring on pathogens with recent updates and their antimicrobial sensitivity pattern is mandatory for the higher management. Objective: This study was designed to evaluate the Bacteriological Profile and Antimicrobial Sensitivity Pattern of Neonatal Sepsis. Methods: This prospective observational study was under taken in the neonatal unit, department of pediatrics, Faridpur Medical College Hospital, Faridpur, Bangladesh from March 2020 to March 2021. Blood samples from 90 clinically suspected neonatal sepses were collected and processed in the microbiological protocol and their antimicrobial sensitivity pattern was determined. Results: Of 90 cases, 61(67.78%) showed positive blood culture. Gram positive isolates were 48(78.79%) and Gram negative isolates were 23(37.71%).Staphylococcus aureus was the most common organism 75.51% followed by E.Coli 27.87%, Klebsiella pneumoniae 9.84% and Streptococcus pneumonia 3,.28% among the isolates. All the three common isolates showed 100% resistance to Ampicillin. Gram positive isolates were sensitive to Amikacin and Amoxicillin & Clavulenic acid whereas Gram negative isolates were sensitive to Gentamicin and Meropenum. Conclusion: Neonatal septicemia was found to be 67.78% during this study supported blood culture because the gold standard investigation for diagnosis. This result also showed that there was an increasing rate of antibiotic resistance to the commonly used antibiotics. These two changes in microbial spectrum and antimicrobial susceptibility pattern as noticed during this study will definitely help in treating such cases with appropriate antibiotic and thereby help to decrease neonatal morbidity and mortality.