Radiogenic dental damage is a common and crucial problem in patients receiving radiotherapy for malignancies in the head and neck region. Unfortunately, little is known about the development of complications after radiation therapy on the microstructure profiles of the human tooth. Therefore, we propose a novel method in which the primary focus is to investigate, in vitro, the direct influences of different radiation doses on elastic properties of enamel and dentin of human tooth by Scanning Acoustic Microscopy (SAM) at the microscale. We obtain two-dimensional (2D) acoustic impedance images from twenty-five sound human third molars each of which is cut into a 1 mm thick cross-sectional slices. Acoustic impedance by SAM operating at 320 MHz are recorded from the sections comprising enamel and dentin before and after every irradiation dose to a cumulative dose of 60 Gy. The findings of our study reveal that radiation therapy changes the micro-elastic features of enamel and dentin accompanied by the decreased acoustic impedance. We establish a relationship between cumulative irradiation doses and the measured acoustic impedance. The quantified acoustic impedance values for the different irradiation doses might be helpful in in vitro assays for the determination of the safe dose limits to prevent severe tooth damage in the treatment plan of the individuals having head and neck cancer.