Direct microscopy, culture based studies and DNA-DNA hybridization have previously demonstrated an association between microorganisms and osteoradionecrosis. The purpose of our study was to use culture independent molecular techniques to detect bacteria in necrotic bone lesions of the mandible after radiotherapy. Bacterial DNA was extracted from six deep medullary specimens from resected mandibles, including one sample of a relapse. 16S rRNA genes were PCR amplified, cloned, transformed into Escherichia coli and sequenced to determine species identity and closest relatives. From the analysis of 438 clones, 59 predominant species were detected, of which 27% have not been cultivated. The predominant species detected from radionecrotic mandibles were Campylobacter gracilis, Streptococcus intermedius, Peptostreptocooccus sp. oral clone FG014, Uncultured bacterium clone RL178, Fusobacterium nucleatum, and Prevotella spp.. The analysis demonstrated intersubject variability of the bacteria present in osteoradionecrosis. In contrast to the diverse bacterial profile detected in primary infection, only a few members of the oral indigenous flora were identified from a case of relapse. Detection of all members of the complex bacterial flora associated with osteoradionecrosis seems to be necessary to better understand the pathogenesis and to improve the therapeutic approach of the infection.