Background: Meningitis is a serious communicable disease with high morbidity and mortality rates. It is an endemic disease in Egypt caused mainly by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. In some settings, bacterial meningitis is documented depending mainly on positive cerebrospinal fluid (CSF) culture results, missing the important role of prior antimicrobial intake which can yield negative culture. Aim of the Work: This study aimed to utilize molecular technology in order to diagnose bacterial meningitis in both culture-negative and culture-positive CSF samples. Subjects and Methods: The current study is a cross sectional descriptive study performed in the Oncology Diagnostic Unit-Research department at Faculty of Medicine-Ain Shams University in collaboration with Pediatrics Department, Faculty of medicine-Al-Azhar University during the period from 1/8/2015 to 1/8/2017; a total of 55 CSF samples for meningitis patients were included collected from Al Mataria teaching Hospital and Abbassia Fever Hospital. CSF samples were examined by traditional culture method and molecular analysis using real-time quantitative polymerase chain reaction (RT-qPCR). Results: Our study revealed that the overall expression for detection of S. pneumoniae, N. meningitidis, and H. influenzae. A total of 15 (27%) samples had a specific etiologic agent identified, with S. pneumoniae (n = 5), N. meningitidis (n = 5), and H. influenzae (n = 5); these results were obtained by culture. However, the detection efficiency of RT-qPCR for identification of bacterial meningitis causative organism was 100% in the three studied microorganisms. 13 cases showed positivity to H.Influenzeae; 13 for N.meningitidis and 29 for S.pneumoniae which was the commonest pathogen in this study.  Conclusion: Referred to these data;RT- qPCR considered a highly efficient test for detection of bacterial pathogen in meningitis patients; its efficiency exceeds traditional culture methods especially in settings with high rates of culture-negative results