OBJECTIVES: Current multiplex assays cannot detect all four WHO priority pathogens for meningitis diagnosis (Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus agalactiae). This work developed a novel real-time PCR assay capable of simultaneously detecting these pathogens. METHODS: Forty-four DNA samples, including type cultures (NCTC and ATCC), were tested. Specific primers and probes targeting sodC, to detect N. meningitidis, dmsA for H. influenzae, SP2020 for S. pneumoniae, and cfb for S. agalactiae were evaluated in monoplex and multiplex. Standard curves were generated, and limits of detection (LLD), slope, intercept, and R² were determined. Sensitivity, specificity, and predictive values (PPV/NPV) were assessed for both monoplex and multiplex assays. RESULTS: Monoplex and multiplex assays showed equivalent performance. Sensitivities were 100% for all targets; specificities ranged 91.7-100%, PPVs 72.7-100%, and NPVs 100%. The multiplex assay showed high efficiency and consistent amplification for each target gene. LLDs ranged from 24 (S. pneumoniae) to 66 (H. influenzae) genome copies/µl. CONCLUSIONS: The multiplex PCR assay showed good performance for rapid and accurate detection of meningitis-associated bacteria. This test has applications for improved diagnosis of meningitis in LMICs, where laboratory confirmation of major pathogens such as Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis and Group B Streptococcus remains limited. Field validation with clinical specimens is required before implementation.