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Early and accurate diagnosis of bacterial meningitis remains a challenge to the clinician because of its rapid lethal course. The common practice of antibiotic therapy prior to lumbar puncture reduces the chance of isolation of bacteria in CSF culture, making the etiological diagnosis difficult. The present study was done to evaluate a multiplex PCR based method for simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. Three species-specific primers were used along with universal primers of bacterial gene 16S rRNA, in a two-stage PCR assay. A cross sectional prospective study was carried out with 140 children (2 months to 12 years of age) with clinical suspicion of meningitis. Of these 140 patients, 42 (30%) cases were diagnosed as bacterial meningitis and other 98 (70%) as viral meningitis. Among the antibiotic users, bacterial meningitis case detection by PCR was higher (65.52%) than that of culture (10.34%) and Gram staining (6.90%). Regarding the advantages and disadvantages of the conventional bacteriological methods and molecular biology techniques, such as PCR, it is recommended to use both the methods in clinical practice.