Otitis media is the most common infectious disease in young children, which results in changes in the thickness and mechanical properties of the tympanic membrane (TM) and induces hearing loss. However, there are no published data for the dynamic properties of the TM in otitis media ears, and it is unclear how the mechanical property changes are related to TM thickness variation. This paper reports a study of the measurement of the dynamic properties of the TM in a chinchilla acute otitis media (AOM) model using acoustic loading and laser Doppler vibrometry (LDV). AOM was created through transbullar injection of Haemophilus influenzae into the middle ear, and AOM samples were prepared 4 days after inoculation. Vibration of the TM specimen induced by acoustic loading was measured via LDV over a frequency range of 0.1–8 kHz. The experiment was then simulated in a finite element (FE) model, and the inverse-problem solving method was used to determine the complex modulus in the frequency domain. Results from 12 ears (six control and six AOM) show that the storage modulus of the TM from AOM ears was on average 53% higher than that of control ears, while the loss factor was 17.3% higher in control ears than in AOM ears at low-frequency (f < 1 kHz). At high-frequency (e.g., 8000 Hz), there was a mean 40% increase in storage modulus of the TM from AOM compared to control samples. At peak frequency (e.g., 3 kHz), there was a 19.5% increase in loss factor in control samples compared to AOM samples. These findings quantify the changes induced by AOM in the chinchilla TM, namely, a significant increase in both the storage and loss moduli.