Background: Neonatal septicemia is a clinical condition characterized by systemic signs and symptoms due to bacteremia in the first month of the life. Neonatal septicemia is considered one of the leading causes of neonatal mortality globally. The risk factors those may be associated with neonatal septicemia are premature rupture of membrane, prolonged rupture, prematurity, urinary tract infection, poor maternal nutrition, low birth weight, birth asphyxia, and congenital anomalies. The spectrum of organisms causing neonatal septicemia shows variation in different countries and even varies in hospitals of the same region. In developed countries, gram-negative organisms are the most common organisms of neonatal septicemia. The present study objective was to study the spectrum of bacterial isolates from blood culture and their sex ratios in causing septicemia in neonates. Materials and Methods: A Total of 140 blood samples were screened for sepsis in new borns less than 28 days old suspected on clinical grounds. 2ml of blood collected and inoculated in 10 ml of Brain Heart Infusion Broth(BHI). Subculture was done on blood agar. The growth from mac conkey or blood agar was noted and the organism was identified by standard characteristic tests, further subculture from BHI was done on 3rd and 7th day of incubation. Result: Among the 140 patients, 26 showed Gram Positive organisms, 52 showed Gram negative organisms, 11 Candida /yeasts like were isolated, and 51 showed No Growth after 3 times subculture. Among the 26 Gram Positive organisms, Staphylococcus aureus were 15(57.69%) and enterococcus were 11 (42.31%). Among the 52 Gram Negative organisms, Escherichia coli were 3(5.76%), Klebsiella pneumonia 23(44.24%) , NFGNB were 14 (26.92%), Citrobacter spp 4(7.68%). Conclusion: Positive blood culture is the gold standard in the diagnosis of neonatal sepsis. Early identification of organisms causing neonatal sepsis and appropriate use of antibiotics to minimize the morbidity and mortality and emerging resistance in bacteria is essential to control neonatal sepsis in neonatal intensive care units.