Introduction: Dentin hypersensitivity (DH) is a very common problem that faces many patients and maintaining treatments with a long term effectiveness remains a challenge. Based on hydrodynamic theory, the main approach of overcoming DH are tubular occlusion. Objectives: The aim of this study was to discover the efficacy of diode laser alone and in combination with a desensitizing toothpaste containing nano-hydroxyapatite (nHap) to evaluate dentinal tubule occlusion after acid challenge under scanning electron microscope (SEM). Material and methods: Forty dentin discs were divided into 4 groups: group 1, control; group 2, dentin disc treated with nHap toothpaste for one week; group 3, discs treated with diode laser in non-contact mode for 60 seconds; group 4, discs treated with nHap toothpaste for one week, followed by laser application. SEM analysis was performed after acid challenge to the discs. Results: Tubular occlusion was evident in all groups after acid challenge; however, group 4 showed the most occluded tubules, which was the most resistant to acid challenge. Conclusions: Combination of diode laser and toothpaste containing nHap has a greater effect on occluding dentinal tubules rather than each treatment alone.