The aim of this study was to investigate the microbial composition of necrotic root canals using culture methods and microarray technology. Twenty uniradicular teeth with radiographic evidence of periapical bone loss and with no previous endodontic treatment were selected for this study. For molecular diagnosis a DNA chip with 20 different species-specific, 16S-rDNA-directed catcher probes was used. The microorganisms most frequently detected by the DNA chip were: Micromonas micros, Fusobacterium nucleatum ssp., Tannerella forsythia, Treponema denticola, Veillonella parvula, Eubacterium nodatum, Porphyromonas gingivalis, Actinomyces odontolyticus, and Streptococcus constellatus. As expected, additional important bacterial taxa were found by culture analysis, but microorganisms such as T. forsythia and T. denticola could not be identified. In conclusion, microarrays may provide significant additional information regarding the endodontic microbiota by detecting bacterial species that are otherwise difficult or impossible to culture.