The tympanic membrane forms an impenetrable barrier between the ear canal and the air-filled middle ear, protecting it from fluid, pathogens, and foreign material entry. We previously screened a phage display library and discovered peptides that mediate transport across the intact membrane. The route by which transport occurs is not certain, but possibilities include paracellular transport through loosened intercellular junctions and transcellular transport through the cells that comprise the various tympanic membrane layers. We used confocal imaging to resolve the phage's path through the membrane. Phages were observed in puncta within the cytoplasm of tympanic membrane cells, with no evidence of phages within junctions between epithelial cells. This result indicates that transport across the membrane is transcellular and within vesicles, consistent with the transcytosis process. The trans-tympanic peptide phages display a wide range of transport efficiencies for unknown reasons. This could include variation in tympanic membrane binding, entry into the membrane, crossing the membrane, or exiting into the middle ear. To address this, we titered phages recovered from within the membrane for phages with differing transport rates. We found that differences in the transport rate were inversely related to their presence within the tympanic membrane. This suggests that differences in the transport rate primarily reflect the efficiency of an exocytotic exit from the mucosal epithelium rather than entry into, or passage across, the membrane.