Introduction: Neonatal sepsis (NS) is one of the important causesof neonatal morbidity anddeath in neonatal ward and neonatal medical care unit,especiallyin emergent nations. Frequent monitoring on pathogens with recent updates and their antimicrobial sensitivity pattern is compulsory for more satisfactory management. Methods: This cross sectionalstudy was outlined to evaluate the bacteriological profile and antimicrobialsensitivity pattern of NS in neonatal ward and neonatal intensive care unit (NICU)of Sylhet Women’s Medical College and Hospital, Sylhet, Bangladesh from October 2019 to December 2020. Blood samples from 90 clinically suspected neonatal sepsis cases were accumulated and processed in the microbiological protocol and their antimicrobial sensitivity pattern were determined. Results: Among the90 cases, two third (61, 67.78%) showed positive blood culture. Among the positive blood culture, 38(62.29%) were gram positive isolates and gram negative isolates were 23(37.7%). This study showed Staphylococcus aureuswas the most common organism (59.01%) followed by Escherichia coli (E. Coli)(27.87%), Klebsiella pneumonia (9.87%) and Streptococcus pneumonia (3.28%) among the isolates. Gram positive isolates were highly reactive to Amikacin and Amoxicillin and Clavulenic acid whereas gram negative isolates were highly reactive to Gentamicin and Meropenem. Conclusion: Gram positive organism are the predominant source of NS which was highlighted in our study,though gram negative organism also plays an important role in the mortality cause by neonatal sepsis.