Rapid and accurate detection of pathogenic bacteria is important for the treatment of patients with suitable antibiotics. Here we report the development of a diagnostic DNA microarray for the high-throughput identification of 39 pathogenic bacteria selected based on their high prevalence rate and/or difficulty of cultivation. The 23S ribosomal DNA and 16S-23S rDNA intergenic spacer region were used as target DNAs for pathogen detection. Universal- and species-specific probes were designed based on the unique and common sites within the target DNA sequences. New target DNA sequences were determined for the detection of 19 bacterial pathogens. The usefulness of the designed probes was validated using 39 reference bacteria and also with 515 clinical isolates from various clinical samples including blood, stool, pus, sputum, urine and cerebrospinal fluid. The DNA microarray developed in this study allowed efficient detection of bacterial pathogens with the specificities of 100%. The sensitivities were 100% as well except for the two pathogens, Enterobacter cloacae (75%) and Enterococcus faecium (85%). These results suggest that the DNA microarray-based assay developed in this study outperforms current diagnostic systems with respect to sensitivity, specificity, and high-throughput detection, and thus should be useful in pathogen diagnosis in the clinical setting.