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Background: Acute bacterial meningitis is among serious infections of the central nervous system (CNS). The early diagnosis and empiric antibiotic treatments have led to a reduction in morbidity and mortality rates. PCR and the enzymatic digestion of 16S rDNA fragment following the PCR by universal primers led up fast and sensitive determination. The aims of the present study was to improve our previous method for rapid and specific detection of common bacteria causing acute meningitis. Methods: According to the gene encoding 16S rDNA found in all bacteria, a set of primers was designed. Then the universal PCR was performed for bacterial agents of meningitis by employing broad-range DNA extraction method. The amplicons were digested with restriction enzymes to identify bacterial species. Results:  By the  enzymatic  digestion  of  the  amplicons  of  each  standard strain, specific patterns were achieved. These specific patterns may be used for comparison in CSF examination. The analytical sensitivity of the assay was  approximately 1.5×102   CFU/ml  of  CSF  even in  samples with  high amount of proteins. Conclusion: The universal PCR coupled with enzymatic digestion can be used to detect and identify bacterial pathogens in clinical specimens rapidly and accurately. Molecular diagnostic of bacterial meningitis, though expensive and labor-intensive, but is valuable and critical in patient management.