ABSTRACTOrofacial granulomatosis (OFG) is a chronic inflammatory disease characterized by nontender swelling of the orofacial tissues, the underlying cause of which remains unknown. Our previous study demonstrated that tooth apical periodontitis (AP) is involved in the development of OFG. To characterize the AP bacterial signatures of OFG patients and identify possible pathogenic bacteria that cause OFG, the compositions of the AP microbiotas in OFG patients and controls were compared using 16S rRNA gene sequencing. Pure cultures of putative bacterial pathogens were established by growing bacteria as colonies followed by purification, identification, and enrichment and then were injected into animal models to determine the causative bacteria contributing to OFG. A specific AP microbiota signature in the OFG patients was shown, characterized by the predominance of phylaFirmicutesandProteobacteria, notably members of the generaStreptococcus,Lactobacillus, andNeisseria, were found.Streptococcusspp.,Lactobacillus casei,Neisseria subflava,Veillonella parvula, andActinomycesspp. from OFG patients were isolated and successfully culturedin vitroand then injected into mice. Ultimately, footpad injection withN. subflavaelicited granulomatous inflammation.IMPORTANCEInfectious agents have long been considered to play a role in the initiation of OFG; however, a direct causal relationship between microbes and OFG has not yet been established. In this study, a unique AP microbiota signature was identified in OFG patients. Moreover, we successfully isolated candidate bacteria from AP lesions of OFG patients and assessed their pathogenicity in laboratory mice. Findings from this study may help provide in-depth insights into the role of microbes in OFG development, providing the basis for targeted therapeutic approaches for OFG.