Background: Neonatal septicemia is a clinical illness that occurs within the first four weeks of life and is defined by systemic signs and symptoms brought on by a widespread bacterial infection. The most frequent cause of illness and mortality in the neonatal era is bacterial infections. Early detection of this potentially fatal illness is essential for prompt treatment and a successful outcome since complications like shock, disseminated intravascular coagulation, and multi-system organ failure can lead to the fulminant and lethal course of infection. Objective: The present study was undertaken to detect the causative organism of neonatal sepsis by blood culture. Method of the Study: This is a cross- sectional study carried out in the Department of Neonatology, BSMMU, Dhaka over a period of 6 months between January 2013 to June 2013. Newborns with suspected sepsis admitted to BSMMU were the study population. Clinically diagnosed cases of neonatal sepsis aged < 28 days of both sexes whose parents or guardians provided informed consent were eligible for enrollment in the study. The subjects were selected consecutively from the study population. Data were collected using a structured questionnaire containing all the variables of interest. Data were processed and analyzed using the computer software SPSS (Statistical Package for Social Sciences). The test statistics used to analyze the data were Chi-square (χ2) or Fisher’s Exact Probability Test & Student’s t-Test. Result: The study included a total of 138 neonates (both preterm and term) of clinically diagnosed cases of sepsis. All the neonates were subjected to test for WBC, IT ratio CRP, and blood culture. In terms of gestational age over 80% were preterm and the rest 19.6% were term neonates. Among the neonates, 40 (29%) had early onset sepsis (EOS) and 98(71%) had late-onset sepsis (LOS). Of the culture- positive cases, Klebsiella was predominant (33.33%) followed by E. coli (22.2%), Acinetobacter (16.66%) and Pseudomonas (11.1%). Less commonly found organisms are Staphylococcus aureus, Enterobacter, Enterococcous Cytrobacter, and others. Conclusion: Sepsis screening is more useful for ruling out the diagnosis of newborn septicemia, which can be made rationally if two screenings conducted 12 to 24 hours apart are negative. It is preferable to wait for the findings of the sepsis screen before starting antibiotics in a neonate who is otherwise stable or suspected of having sepsis due to maternal risk factors.