Our work proposes a novel approach to investigate bacterial biofilms in human middle ear using two methods, optical coherence tomography (OCT) and acoustic measurements. Noninvasive OCT images were collected to visualize cross-sectional structure of the middle ear and confirm the presence of biofilms behind the tympanic membrane (TM). The acoustic measurements implemented by multi-frequency power reflectance provide sound reflectance/transmittance/impedance characteristics of the middle ears. These acoustic measurements are then represented by poles and zeros plots which are then analyzed to determine physical acoustic characteristics and to synthesize a new acoustic model of the middle ear. This method combination of optical imaging and acoustic measurements is implemented on normal and pathologic human ears to study the presence of biofilms and their impact on conductive hearing loss.