In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene (rpoB) for detecting 42 oral bacterial species. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 73-79 strains regarding 73-75 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of 42 bacterial species-specific qPCR primers ranged from 4 to 40 fg below a cycle threshold (C T) value of 35, except Atopobium rimae, Fusobacterium nucleatum, Neisseria meningitidis, and Porphyromonas asaccharolytica which were 400 fg. These results suggest that 42 bacterial species-specific qPCR primers are suitable for applications in epidemiological studies related to oral infectious diseases such as periodontal diseases, endodontic infection, and dental caries.