Material and Methods: In this study a total of 173 specimen were cultured including peripheral samples 160 (92.5%), tips of endotracheal tubes 8 (4.6%) and tracheal aspirates 5 (2.8%). Our patients were newborns age ranging from 5 -19 days who were admitted due to suspected septicemia. Specimens were sent to Shaukat Khanam Memorial Hospital Lahore and Aga Khan University Hospital, Karachi where they were cultured by using standard microbiological methods. The necessary information's regarding age of the neonates, weight, gestational age, mode of delivery, prior use of antibiotics and outcome were recorded on predesigned data collection sheet. Results were analyzed by using statistical tools. Results: The outcome of 173 specimens, microbial testing showed that 91 (53%) isolates were found to be positive. Out of these 91, 77 (85%) were Bacterial and 14 (15%) were fungal species. The incidence of Gram Negative and Gram Positive bacteria was found to be 62 (68%) and 15 (16%) respectively. The most common gram negative specie was Klebsiella Pneumonia 20 (32%). Among gram positive bacteria staphylococcus epidermidis 15 (67%) was the most common species. Among fungal species Candida sepsis was the most common, 13 (14%) of all the samples. Most of the gram negative organisms were sensitive to Aminoglycosides, Imipenem and Ciprofloxacin. Gram positive species were sensitive to Linezolid and Vancomycin. All the fungal species were sensitive to Fluconazole and Voriconazole. Conclusion: There is a variety of organisms causing neonatal septicemia in Tertiary Care hospitals with variable susceptibility pattern, which reflects the need to evaluate the incidence of different microbial species after every quarter and study there susceptibility patterns.