This study aimed to evaluate the influence of radiotherapy and different endodontic treatment protocols on the bond strength to pulp chamber dentin. Eighty mandibular molars were randomly divided into two groups (n = 40): non-irradiated and irradiated (60Gy). The pulp chambers were sectioned, and each group was subdivided (n = 8), according to the endodontic treatment protocol: no treatment (Control); Single-visit; Two-visits; Immediate dentin sealing (IDS) + single-visit; and IDS + two-visits. Each endodontic treatment visit was simulated through irrigation with 2.5% NaOCl, 17% EDTA and distilled water. IDS was performed by actively applying two coats of a universal adhesive to the lateral walls of the pulp chamber. After, the pulp chambers were restored with resin composite and four sticks were obtained for microtensile test. In addition, the dentin of the pulp chamber roof was assessed for surface roughness, chemical composition, and topography after each treatment protocol. Two-way ANOVA, Tukey's post hoc, Mann-Whitney, Kruskal-Wallis and Dunn's post hoc were performed (α = 5%). The treatment protocol affected bond strength (p < 0.05), while the irradiation did not (p > 0.05). The control group presented the highest values (p < 0.05). The single-visit group demonstrated better performance compared to the other groups (p < 0.05), which did not differ from each other (p > 0.05) The use of IDS changed the surface roughness (p < 0.05), chemical composition (p < 0.05) and topography of the dentin. In conclusion, the treatment protocol influenced dentin adhesion, while irradiation did not.