Central nervous system infection (CNSI) results in significant health and economic burdens worldwide, but the diversity of causative pathogens makes differential diagnosis very difficult. Although PCR and real-time fluorescent quantitative PCR (q-PCR) assays are widely applied for pathogen detection, they are generally optimized for the detection of a single or limited number of targets and are not suitable for the diagnosis of numerous CNSI agents. In this study, we describe the development of a resequencing pathogen microarray (RPM-IVDC4) method for the simultaneous detection of viruses, bacteria, fungi and parasites that cause CNSI. The test panel of this assay included more than 100 microorganism species across 45 genera and 30 families. The analytical specificity and sensitivity were examined using a panel of positive reference strains, and the clinical performance was evaluated using 432 clinical samples by comparing the results with q-PCR assays. Our results demonstrated good performance of the RPM-IVDC4 assay in terms of sensitivity, specificity and detection range, suggesting that the platform can be further developed for high-throughput CNSI diagnosis.