Background: Bacterial meningitis is more common in the neonatal period than any other time in life; however, it is still a challenge for the evidence based diagnosis. Strategy for identification of neonatal bacterial meningitis pathogens is presented by evaluating three different available methods to establish evidence-based diagnosis for neonatal bacterial meningitis. Methods: The cerebrospinal fluid samples from 56 neonates diagnosed as bacterial meningitis in 2009 in Beijing Children's Hospital were analyzed in the study. Two PCR based molecular assays, real-time fluorescence quantitative PCR (RT-PCR) and multiplex PCR based-reverse line blot hybridization (mPCR/RLB), were used to assess 7 common neonatal meningitis bacterial pathongens, including Escherichia coli, Staphylococcus aureus, Listerisa monocytogenes, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, and Streptococcus agalactiae. The findings in examinations of two assays were compared with the results obtained bacterial culture tests. Results: Bacterial meningitis was identified in five cases (9%) by CSF cultures, 25 (45%) by RT-PCR and 16 (29%) by mPCR/RLB. One strain of S. epidermidis and one of E. faecalis were identified using mPCR/RLB but not by RT-PCR. In contrast, cultures identified one strain of S. pneumoniae which was missed by both PCR assays. Overall, the bacterial pathogens in 28 cases were identified with these three methods. Both RT-PCR and mPCR/RLB assays were more sensitive than bacterial culture, (p < 0.05). Conclusion: Our study confirmed that both RT-PCR and mPCR/RLB assays have better sensitivity than bacterial culture. They are capable of detecting the pathogens in CSF samples with negative culture results.