Introduction: Neonatal sepsis is one of the significant causes of mortality and morbidity in neonatal age. Prompt clinical diagnosis and empirical antibiotic therapy is essential to decrease the mortality and morbidity. Because the causative organisms and their susceptibility are different, place to place and time to time, and resistant to commonly used antibiotics, it is alarming all over the world especially in developing countries Therefore, periodic causative and antimicrobial surveillance is essential all over the world. Objective: The aim of this study was to determine the frequency of causative organisms and their susceptibility to commonly used antibiotics in neonatal sepsis as well as to direct the empirical antibiotic therapy at French Medical Institute for Children (FMIC), Kabul Afghanistan. Material and Methods: A descriptive cross sectional study was conducted. A total of 270 blood culture positive patients were included in the study from January 2013 to June 2016. Medical record files and blood culture results were reviewed. Results: A total of 1184 suspected neonatal sepsis patients were admitted. Blood culture was positive in 298 (25.1%), of which 172 (63.7%) were gram negative; while 90 (33.3%) were gram positive organisms and 8 (2.9%) were with fungal growth. Pseudomonas 42 (15.5%) and Klebsiella 41 (15.1%) were the leading causes among gram negative organisms followed by Burkholderia (10.7%), Enterobacter cloacae (7.7%), E. coli(5.5%) and other gram negative organisms. CoNS 68(25.1%) was the most common organism among gram positive following by Staphylococcus aureus 15 (5.5%) and Enterococcus 7 (2.6%). All isolated organisms were highly resistant to Ampicillin (>75%), Cefotaxime (>50%) and Gentamycin (>25%), while the most effective drug for gram negative organisms were Imipenem, Tazobactam+Piperacillin, Amikacin and Ciprofloxacin. All gram positive organisms were highly sensitive (100%) to Vancomycin. Conclusion: Gram negative organisms were the leading cause of neonatal sepsis followed by gram positive. All isolated organism were highly resistant to commonly used empirical antibiotics for neonatal sepsis. Irrational use of antibiotics by physicians is leading to resistance, mortality and morbidity. In order to prevent all these bad outcomes, regular educational awareness programs in the hospitals and regular surveillance for detecting the common causes and their susceptibility is crucial to adapt the empirical antibiotic therapy in neonatal sepsis.