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To characterize otitis media–associated structures affixed to the mucosal surface of the tympanic membrane (TM) in vivo and in surgically recovered in vitro samples.Prospective case series without comparison.Outpatient surgical care center.Forty pediatric subjects scheduled for tympanostomy tube placement surgery were imaged intraoperatively under general anesthesia. Postmyringotomy, a portable optical coherence tomography (OCT) imaging system assessed for the presence of any biofilm affixed to the mucosal surface of the TM. Samples of suspected microbial infection–related structures were collected through the myringotomy incision. The sampled site was subsequently reimaged with OCT to confirm collection from the original image site on the TM. In vitro analysis based on confocal laser scanning microscope (CLSM) images of fluorescence in situ hybridization–tagged samples and polymerase chain reaction (PCR) provided microbiological characterization and verification of biofilm activity.OCT imaging was achieved for 38 of 40 subjects (95%). Images from 38 of 38 (100%) of subjects observed with OCT showed the presence of additional microbial infection–related structures. Thirty-four samples were collected from these 38 subjects. CLSM images provided evidence of clustered bacteria in 32 of 33 (97%) of samples. PCR detected the presence of active bacterial DNA signatures in 20 of 31 (65%) of samples.PCR and CLSM analysis of fluorescence in situ hybridization–stained samples validates the presence of active bacteria that have formed into a middle ear biofilm that extends across the mucosal layer of the TM. OCT can rapidly and noninvasively identify middle ear biofilms in subjects with severe and persistent cases of otitis media.