In molecular diagnosis of infectious diseases often more than 1 pathogen has to be considered. As a consequence, a series of labor-intensive and time-consuming polymerase chain reaction (PCR) approaches specific for different putative pathogens have to be carried out. To speed up diagnosis, we established a low-density microarray for simultaneous detection of diverse putative pathogens causing a disease such as granulomatous lymphadenitis. Nucleic acids from formalin-fixed, paraffin-embedded tissues of 68 patients with lymphadenitis were used for molecular diagnosis of individual pathogens by either nested single-assay PCR or 1-step multiplex PCR in combination with low-density microarray hybridization. Multiplex PCR amplicons hybridized to glass slides containing probes from Mycobacterium spp., Yersinia spp., Bartonella henselae, Toxoplasma gondii, and other pathogens showed specific and reproducible signals on the array. Our results show that microarray technology combined with multiplex PCR is a promising and time-saving tool in molecular pathology of infectious diseases, allowing sensitive, simultaneous analyses of different pathogens.