Background On the elastic profiles of human teeth after radiotherapy for head and neck cancers, generation of dental complications, which may bring several side effects preventing the quality of life, has not well clarified. Thus, we aimed to show the applicability of using 320 MHz Scanning Acoustic Microscopy (SAM) in the evaluation of the tooth damage acoustically at the micrometer level following radiation therapy, and also in the determination of the safe dose limits to impede severe dental damage. Methods This prospective study was performed by SAM employed at 320 MHz by an azimuthal resolution of 4.7 mu m resolving enamel and dentin. A total of 45 sound human third molar teeth collected between September 2018 and May 2019 were used for the acoustic impedance measurements pre- and post irradiation. Nine samples for each group (control, 2 Gy, 8 Gy, 20 Gy, 30 Gy and 60 Gy) were evaluated to acquire the acoustic images and perform a qualitative analysis. Scanning Electron Microscopy (SEM) images were obtained to establish a relationship between micromechanical and morphological characteristics of the teeth. Statistical analysis was conducted using the Student t-test succeded by Mann-Whitney U investigation (p < .05), while SEM images were assessed qualitatively. Results The analysis included 45 sound teeth collected from men and women 18 to 50 years old. Post irradiation micromechanical variations of human teeth were significant only in the radiation groups of 30 Gy and 60 Gy compared to pre-irradiation group for enamel (7.24 +/- 0.18 MRayl and 6.49 +/- 028 MRayl; p < 0.05, respectively). Besides, the teeth subjected to radiation doses of 20, 30 and 60 Gy represented significantly lower acoustic impedance values relative to non-irradiated group for dentin (6.52 +/- 0.43 MRayl, 5.71 +/- 0.66 MRayl and 4.82 +/- 0.53 MRayl p < 0.05), respectively. Conclusions These results are evidence for a safe acoustic examination device which may be a useful tool to visualize and follow the safe dose limits to impede severe dental damage through the radiation therapy treatment for head and neck cancers.