The objective of this study was to determine the effects of various irrigation solutions on root canal dentine and gutta-percha surface properties. In addition, the effects of disinfectant chemicals on the wettability and surface morphological properties of the filling materials were evaluated. Ethylenediaminetetraacetic acid (EDTA), citric acid, and ozone were employed as irrigation solutions for dentine and gutta-percha treatment. Thereafter, the samples’ microstructure, degree of mineralization, and mechanical properties were assessed by means of quantitative backscattered electron imaging (qBEI) and reference point indentation (RPI). A contact angle analyzer was used to measure adhesion on the tested materials. Here, EDTA had the most significant affect on both the mechanical properties and the adhesive behavior of dentine. Citric acid did not affect dentine wettability, whereas the indentation properties and the mineralization were reduced. Similar effects were observed when ozone was used. The dentinal tubules were significantly widened in citric acid compared to the ozone group. EDTA causes considerable micromechanical surface alteration of dentine and gutta-percha, but represents the best option in clinical cases where a high adhesiveness of the filling materials is desired.