Acute otitis media (AOM) is a rapid onset of infection in the middle ear and the most frequently diagnosed disease in pediatric population. Conductive hearing loss is the most prevalent outcome of AOM because pathological changes in the infected middle reduce mobility of the tympanic membrane (TM). Mechanisms of TM mobility loss associated with AOM are not well understood. We hypothesize that middle ear pressure (MEP), middle ear effusion (MEE), and structural changes of ossicular adhesions and ear soft tissues are the main factors contributing to the loss of TM mobility in AOM ears and their effects vary during the course of the disease. In this dissertation, a chinchilla AOM model was produced by transbullar injection of Haemophilus influenzae. Changes of MEP, MEE, and ossicular adhesions were characterized at day 4 (4D) and day 8 (8D) post inoculation. These time points represent relatively early and later phases of AOM. Microstructural changes of the TM, round window membrane (RWM), and stapedial annular ligament (SAL) in the early and later phases of AOM were investigated by histology. Hearing loss in both AOM phases was evaluated by auditory brainstem response (ABR). TM mobility at the umbo and middle ear energy absorbance (EA) were measured in 4D and 8D AOM ears. In each group, the vibration of the umbo and EA was measured at three experimental stages: unopened, pressure-released, and effusion-removed ears. The effects of MEP and MEE and middle ear structural changes were quantified in each group by comparing the TM mobility or EA at one stage with that of the previous stage. Our findings show that the factors affecting TM mobility change with the disease time course. The MEP was the dominant contributor to reduction of TM mobility in 4D AOM ears, but showed little effect in 8D ears when MEE filled the tympanic cavity. MEE was the primary factor affecting TM mobility loss in 8D ears, but affected the 4D ears only at high frequencies. After the release of MEP and removal of MEE, residual loss of TM mobility was seen mainly at low frequencies in both 4D and 8D ears, and was associated with ossicular adhesions. The effects of MEP and MEE on EA were similar to those on TM mobility. Residual loss of EA seems was more likely associated with mechanical property changes of TM. Our findings demonstrate that MEP, MEE, together with middle ear structural changes, contribute to TM mobility loss in the chinchilla AOM ears. Their effects on TM movement at early and later phases of the disease were quantified. This study provides useful experimental data for understanding the mechanism of conductive hearing loss in AOM.