5573 Background: Recent studies of the aerodigestive tract have uncovered the functional importance of the microbiome, the total collection of microorganisms that reside within the human host. Furthermore, specific bacterial species have been shown to be etiologic agents or show potential as a screening biomarker in cancers including gastric, colon and pancreas. Here, we hypothesize that specific microbial populations may contribute to HNSCC pathogenesis via epigenetic modifications in inflammatory- and HNSCC-associated genes. Methods: Matched tumor and adjacent normal fresh frozen tissue specimens were collected from 55 prospectively enrolled HNSCC patients. Microbiomic profiles were obtained using Sanger sequencing of 16S rDNA PCR products. Methylation status of four genes previously linked to HNSCC or inflammation (MDR1, IL8, RARB, TGFBR2) was assessed in 49 samples by combined bisulfite restriction analysis and by Illumina HumanMethylation27 chip. Principle component analysis and multivariate analysis of covariance (MANCOVA) were used to identify bacterial subpopulations significantly associated with HNSCC and relevant clinical variables. Results: In this cohort, median age is 62, male:female ratio is 3:2, 16% are HPV+. Specific bacterial subpopulations associate with HNSCC over normal tissue and correlate with larger tumor size and higher nodal stage (p<0.05). Furthermore, microbial populations can separate tumors by tobacco (p<0.005) but not alcohol (p>0.7) status. Bacterial profiles are independent of HPV status. MDR1 promoter methylation is seen in tumor samples but not in normal oral mucosa (22/49 vs 0/49), and associates with specific microbiomic subpopulations in the order Enterobacteriales and phylum Tenericutes (p<0.001). Additionally, MDR1 methylation correlates with regional nodal metastases. Conclusions: Specific bacterial subpopulations differ between normal and tumor tissue and show association to specific clinicopathologic features, suggesting a role for microbial profiling in HNSCC as a novel area of investigation for diagnosis, prognostication, and therapeutic targeting.