We here report on the development of a novel multiplex PCR with product detection in a Luminex 100 suspension array system. The assay covers the nine most important bacterial and viral pathogens found in Danish meningitis patients. The microorganisms include Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Streptococcus agalactiae, herpes simplex virus types 1 and 2, and varicella-zoster virus. The study was based on 1,187 samples, of which 55 were found to be positive by PCR. The assay was found to have an excellent sensitivity and an excellent specificity compared to the results of a "gold standard," defined by routine laboratory tests, for the two most important pathogens, S. pneumoniae (95 and 99.1%, respectively) and N. meningitidis (100 and 99.7%, respectively). The method provides a valuable supplement to the traditional microscopy and culture of cerebrospinal fluid (CSF) samples in a routine diagnostic setting, and results can be available within 1 workday. The method is suitable for use for the initial screening and identification of nine important microorganisms in CSF samples from patients with suspected meningitis. Compared to microscopy and culture of CSF, this rapid and sensitive method will support physicians with the selection of the appropriate antimicrobial agents and the initiation of timely treatment in the absence of live microorganisms in the CSF.