In the present research, nanoindentation, atomic-force microscopy and optical microscopy were used to study the mechanical and microgeometrical parameters of tooth tissues. A nanoindentation test unit equipped with Berkovich indenter was used to determine the values of the reduced Young’s modulus and indentation hardness and both nanoindentation and atomic force microscopy using a diamond probe on a silicon cantilever were used to study microgeometrical parameters of human tooth root cementum. Three areas of cementum were studied: the cervical region near the dentine–enamel junction, the second third of the tooth root, and the apex of the tooth root. The interpretation of the results was carried out using the Oliver–Pharr method. It was established, that the mechanical properties of cementum increase from the cervical region to the central part of the root, then decrease again towards the apex of the tooth root. On the contrary, the microgeometrical characteristics of cementum practically do not demonstrate any change in the same direction. A decrease in the roughness parameters in the direction from cellular cementum to dentine was observed. Additionally, a decrease in the reduced Young’s modulus and indentation hardness of dentine in the cervical area compared to dentine in the crown part of the tooth was found using nanoindentation. The investigation of the dentine–cementum junction with high resolution revealed the interspaced collagen fiber bridges and epithelial rests of Malassez, whose sizes were studied. The parameters of the topographic features of the cementum in the vicinity of the lacunae of cementocytes were also established.