Background: Dentin hypersensitivity is an important clinical problem affecting a large percentage of the population, and various therapies have been suggested for its treatment—among them desensitizing agents and lasers. The aim of this study was to evaluate the in vitro effects of different in-office desensitizing agents and different type lasers, alone or in combination, on human dentinal tubules. Materials and Methods: Eighty-four dentinal specimens obtained from freshly extracted impacted third molars were included and subsequently divided into seven groups: Group 1 (Control), Group 2 (Gluma desensitizer), Group 3 (Teethmate desensitizer), Group 4 (Nd:YAG laser), Group 5 (Er:YAG laser), Group 6 (Gluma desensitizer + Er:YAG laser), and Group 7 (Teethmate desensitizer + Er:YAG laser). The dentinal specimens of all groups were evaluated using SEM. A total of 12 images per sample were taken, and the numbers of open, partially occluded, and totally visible (open + partially occluded) dentinal tubules were counted. AFM was used to further substantiate the blocking mechanism. The data were analyzed by one-way ANOVA and Dunnett-C tests. Results: Comparison of the number of occluded dentinal tubules per 100 µm2 revealed that the laser and laser combined-treated groups showed the most occluded dentinal tubules. Gluma desensitizer + Er:YAG laser combination demonstrated significantly more tubule occlusion than all the other groups (p <.05). Conclusions: The results of this study show that combined treatment methods were effective in the occlusion of dentinal tubules.