The efficacy of treatment of central nervous system infection depends on the accuracy of the etiologic diagnosis. This article reviews appropriate specimen collection, specimen processing, and microbiology test utilization. In addition, new and evolving microbiology laboratory methods used to detect infected cerebrospinal fluid infections, tuberculosis meningitis, Whipple's disease involving the central nervous system, viral encephalitis, fungal meningitis, and Creuztfeldt-Jakob disease are discussed.