Infections of the central nervous system (CNS) can lead to severe outcomes if not accurately diagnosed and treated. The broad spectrum of pathogens involved in CNS infections can make diagnosis challenging. Polymerase chain reaction (PCR) -based multiplex molecular diagnostic panels can rapidly and simultaneously detect multiple neuropathogens in cerebrospinal fluid (CSF). This study was aimed to assess the Bio-Speedy Meningitis/Encephalitis RT-PCR MX-17 panel (Bioeksen, İstanbul, Türkiye), a novel multiplex PCR test, in diagnosing CNS infections. The panel can detect a range of pathogens, including Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, enterovirus (EV), herpes simplex virus (HSV) 1 and 2, HHV-6, HHV-7, HHV-8, human parechovirus (HPeV), varicella zoster virus (VZV), cytomegalovirus (CMV) and Cryptococcus gatti/neoformans in CSF samples. This retrospective study included 128 CSF samples from 128 patients sent to Bursa Uludağ University Health Application and Research Center Microbiology Laboratory between June 2022 and July 2023 to search for CNS infectious agents. Patient clinical, radiological and laboratory data were collected from the Hospital Information Record System (HIRS). Bacterial pathogens were identified through culture, while viral pathogens were detected in CSF samples using the Fast Track Diagnostics (FTD) multiplex RT-PCR panel (Fast Track Diagnostics Ltd., Luxembourg) for HSV-1, HSV-2, VZV, EV, mumps virus and HPeV. The stored CSF samples were then tested using the BioSpeedy panel and the results were compared with those of the culture and the FTD panel. Pathogens that were detected were considered positive if they were consistent with the patient's symptoms and CSF characteristics according to infectious disease and pediatric infectious disease specialists. Pathogens detected but not supported by the patient's symptoms and CSF characteristics were classified as uncertain clinical relevance (UCR). Out of the 128 patients tested for CNS infectious agents, 44 (34.4%) were diagnosed with a CNS infection. The overall pathogen detection rate with all methods was 43.2% (19/44). The Bio-Speedy panel identified pathogens in 29.5% (13/44) of the patients, followed by the FTD panel (20.5%, 9/44) and culture (9.1%, 4/44). Four bacteria were identified with culture, three of which were also detected by the Bio-Speedy panel. Additionally, six bacteria were identified with Bio-Speedy panel, that were not identified by culture. The FTD panel identified nine viruses, four of which were also identified by Bio-Speedy. In total, the Bio-Speedy panel detected 13 of the 19 positive pathogens (nine bacteria and four viruses: [S.pneumoniae (n= 3), VZV (n= 3), N.meningitidis (n= 2), H.influenzae (n= 2), L.monocytogenes (n= 1), E.coli (n= 1) ve EV (n= 1)]. However, the Bio-Speedy panel identified 15 pathogens [S.pneumoniae (n= 1), E.coli (n= 1), C.gatti/neoformans (n= 1), CMV (n= 8), HHV-6 (n= 3) ve HHV-7 (n= 1)] considered as UCR. The Bio-Speedy identified the causative pathogens in the highest percentage (29.5%) of patients with confirmed CNS infections. Nevertheless, test results should be interpreted based on patient characteristics to ensure appropriate patient management. Using multiple methods and multiplex tests may improve diagnostic accuracy for CNS infections.