Abstract Background: Clinical observations and epidemiologic studies suggest that the incidence of head and neck squamous cell carcinoma (HNSCC) correlates with dental hygiene, implying a role for bacteria-induced inflammation in its pathogenesis. Differences in microbiota in HNSCC compared to matched normal oral mucosa, and whether these differences are accompanied by epigenetic modifications in selected HNSCC-associated genes and clinico-pathologic features has not yet been investigated. Standard microbiologic culturing techniques miss many fastidious organisms. Methods: Human genomic DNA and associated microbial DNA were isolated from 42 prospectively accrued consecutive HNSCC tumors and paired normal oral mucosae. Microbiomic profiling was carried out by serially characterizing bacteria-specific 16S rRNA and taxa classified by the presence of specific polymorphisms in the 16S rDNA genes. Promoter methylation and microbial populations of each sample was determined. Microbiomic profiles were compared between HNSCC and matched normal mucosa, to specific promoter methylation in four genes (MDR1, IL8, RARB, TGFBR2) previously linked to HNSCC or inflammation, and to clinico-pathologic features. Results: Specific microbiomic profiles significantly associate with HNSCC over normal mucosae. While methylation of both MDR1 (p<10-9) and IL8 (p<0.02) genes significantly associate with HNSCC, only MDR1 methylation associates with specific microbiomic profiles in HNSCC over normal mucosae. Furthermore, MDR1 methylation correlates with regional nodal metastases in the context of two specific bacterial taxa, Enterobacteriaceae and Tenericutes. Additionally, microbial populations can separate tumors by tobacco status (p<0.005), but not by alcohol status (p=0.37). Conclusions: Specific microbial populations may contribute to HNSCC pathogenesis, possibly by triggering inflammation and consequent aberrant DNA methylation. These associations could lead to better understanding of the pathogenesis and treatment of HNSCC, suggesting novel roles for demethylating agents and probiotic adjuncts particularly for patients with advanced or refractory disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4087. doi:1538-7445.AM2012-4087