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Background and Objectives: Prompt and accurate diagnosis of acute bacterial meningitis (ABM) is critical for patient management. We designed and evaluated two sets of multiplex-PCR assays for the simultaneous detection of six major etiologies of ABM i.e., Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis in one set and Listeria monocytogenes, Streptococcus agalactiae, and Escherichia coli in another set of multiplex-PCR in CSF of patients with suspected ABM., Methods: A total of 113 CSF specimens from patients of all ages having clinical features suggestive of meningitis were tested for bacteriological evidence by Gram's smear, culture, and our designed multiplex-PCR., Results: Multiplex-PCR assay performed excellently by increasing the overall detection rate by 6% when compared to culture as of total 113 samples tested, 17 (15%) were positive by multiplex-PCR whereas only 9% (10/113) were positive by culture. It detected the DNA in eight culture negative samples revealing the presence of S. pneumoniae in three and other possible bacterial pathogens in five of them. Our assay showed a DNA detection limit of 1 pg/μL. Compared to CSF culture, the sensitivity and specificity of the multiplex-PCR were 90% and 92.2%, respectively., Conclusion: This study accentuates the importance of multiplex-PCR assay that is efficiently fast and reliable for the diagnosis of acute bacterial meningitis that can substantially improve the diagnosis in culture negative cases, especially in patients who were previously started on antimicrobial therapy., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Family Medicine and Primary Care.)