Resistance to antibiotics is highly prevalent in bacterial isolates worldwide, particularly in developing countries.Generally, bacteria acquire antibiotic resistance by genetic changes in the bacterial chromosome or by acquiring extra chromosomal DNA, such as plasmids.Through this study I have tried to evaluate the prevalence and antibiotic sensitivity pattern of E. coli and Klebsiella pneumoniae isolated from the gastrointestinal tract of hospitalized neonates. I have isolated these two pathogen sp. E. coli and K. pneumoniae and confirmed their identity through biochemical tests and API system. I have then characterized the E. coli strains with PCR, to determine whether they belong to the virulent B2 group. Plasmid profile of the E. coli strains has also been determined to understand better the genetic make up of this pathogen that might confer antibiotic resistance.I hope researchers and professionals in the relevant medical/clinical fields should find the data valuable in the context of their own clinical studies.