The present study prepared 2 types of DNA diagnostic chips based on 16S ribosomal DNA (rDNA) and 185-285 rDNA, and evaluated their values in the detection of pathogens in intracranial bacterial/fungal infections. A total of 14 probes of bacteria (Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Haemophilus influenza, Stenotrophomonas maltophilia, Neisseria meningitidis, Enterobacter spp., Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumonia and coagulase negative staphylococcus) and 4 probes of fungi (Candida albicans, Candida tropicalis, Candida glabrata and Cryptococcus neoformans), determined frequently in cerebrospinal fluid (CSF), were designed and used for preparation of microarrays. CSF samples from 88 patients with clinically suspected intracranial infection and standard strains were used to evaluate the chips. The same samples were also analyzed by culture and sequencing. The results demonstrated that the sensitivity, specificity and false-positive rate of the microarray assay compared with culture method were 100 vs. 68.3% (P<0.05), 97.1 vs. 100%, and 2.9 vs. 0%, respectively. The minimum concentration of detection with the chips was 10 cfu ml(-1) for bacteria and 100 cfu ml(-1) for fungi. The specificity of the probes was confirmed, and no cross-reaction was detected in the present study. Furthermore, 13 cases were positive in the microarray and negative in culture. However, 4 cases were not identified as clear pathogens and only positive in the 16S probe sites. The diagnostic DNA microarray for intracranial infections has proven to be more rapid and sensitive, and it may be a better option for clinical application than culture methods.