Background: Septicemia in neonates refers to generalized bacterial infection documented by a positive blood culture in a first 4 weeks of life & is one of the four leading causes of neonatal mortality in India. The emergence of resistant bacteria makes it a requisite to know the prevailing antibiotic susceptibility pattern of the pathogens causing neonatal septicemia Objective: To isolate and identify the etiological agents of Neonatal septicemia at Jawahar Lal Nehru Hospital, Ajmer & to determine drug resistance profile of isolated organisms. Material and methods: Total no. of 355 neonates from J.L.N Hospital clinically suspected of septicemia were studied and out of them 166 were found blood culture positive. Screening and confirmatory test for isolates were done according to clinical laboratory standard institute’s guidelines Result: Out of 355 neonates suspected of septicemia 166 were found blood culture positive. The micro-organisms isolated were gram positive 53.61%, and gram negative 46.38%.Most predominant isolate was Klebsiella pneumoniae (25%) from all the isolated organisms. Methicillin resistance was seen in only 56.6% (17) of COPS (MRSA). (ESBL) producing gram negative organism was seen only in 17% (13). Imipenem was found to be 4% (3) resistant in gram negative isolates. Conclusion: Routine detection of neonatal septicemia is required to be done by each laboratory by the standard detection methods so as to control the spread of these infections. Increase in antibiotic resistance necessitated monitoring of organism susceptibility towards antibiotic.