A primary etiological factor underlying chronic middle ear disease is an inability to open the collapsible Eustachian tube (ET). However, the structure-function relationships responsible for ET dysfunction in patient populations at risk for developing otitis media (OM) are not known. In this study, three-dimensional (3D) finite element (FE) modeling techniques were used to investigate how changes in biomechanical and anatomical properties influence opening phenomena in three populations: normal adults, young children and infants with cleft palate. Histological data was used to create anatomically accurate models and FE techniques were used to simulate tissue deformation and ET opening. Lumen dilation was quantified using a computational fluid dynamic (CFD) technique and a sensitivity analysis was performed to ascertain the relative importance of the different anatomical and tissue mechanical properties. Results for adults suggest that ET function is highly sensitive to tensor veli palatini muscle (TVPM) forces and to periluminal mucosal tissue (PMT) elasticity. Young children and cleft palate subjects exhibited reduced sensitivity to TVPM forces while changes in PMT stiffness continued to have a significant impact on ET function. These results suggest that reducing PMT stiffness might be an effective way to restore ET function in these populations. Varying TVPM force vector relationships via changes in hamulus location had no effect on ET opening in young children and cleft palate subjects but did alter force transmission to the ET lumen during conditions of elevated adhesion. These models have therefore provided important new insights into the biomechanical mechanisms responsible for ET dysfunction.