Background Radiotherapy, applied to the head and neck region, can cause radiation side effects such as reduction of saliva and radiation caries. The aim of this study was to perform an in vitro assessment of the effects of radiation therapy on the morphological, mechanical, and chemical properties of primary and permanent teeth. Methods One hundred four extracted human teeth (52 impacted wisdom teeth, 52 primary molar teeth) were used. The teeth were divided into two parts in the mesiodistal direction. Of the 98 teeth, the vestibular sections were used for the vickers analysis and lingual sections were used for the Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) analysis. The teeth in the experimental group were fixed to wax models. Each model had an equal number of teeth (n = 7). The doses were applied to the teeth for 6 weeks; 5 week days and 2Gy daily. After the radiotherapy was conducted weekly, a wax model was taken from radiation reception. Along with the elemental contents (Na, K, Mg, P, and Ca) of the teeth, enamel and dentin microhardness was evaluated, and SEM analyzes were performed on 6 teeth. Results Radiation caused a decrease in microhardness of enamel and dentin (p < 0.05). In the elemental analysis by ICP-OES, it was observed that there were decreases in all elements after 60Gy compared to the control group (p < 0.05). In the experimental groups, amorphous structures were encountered in SEM images. Conclusions Radiation has negative effects on the teeth structure and additional studies are needed in this regard. This study indicates that radiotherapy patients are at a higher risk for dental caries.