Background Dentinal hypersensitivity is an enigmatic problem that has long bewildered the dental profession and is one domain where man has not yet achieved the elusive “gold standards” of management. Recently, lasers have been used owning to their various modes of action to reduce hypersensitivity. Thus, the aim of the present in vitro study is to evaluate and compare the effect diode laser along with and without Nanocrystalline Hydroxyapatite powder on occlusion of dentinal tubules under scanning electron microscope (SEM). Methodology Twenty specimens were obtained from dentin discs of 2 mm thickness prepared from the cemento-enamel junction (CEJ) portion of 20 extracted human third molar teeth. The specimens were divided in to four groups of 5 specimens each. The specimens were etched in 37% phosphoric acid for 60 seconds to simulate hypersensitivity condition. Specimens in Group 1 (control) received no further treatment. Group 2 (DL) specimens received irradiation with 810 nm diode laser at an output power of 1 W for 60 seconds in continuous wave, non-contact mode. Specimens in Group 3 received treatment with a single application of nanocrystalline hydroxyapatite powder alone, whereas, Group 4 received a combination treatment of Diode laser irradiation followed by application of nanocrystalline hydroxyapatite powder. Results The average tubular diameter of dentinal tubules observed in the specimens belonging to Group 1 (control) was 3.14 microns, and in the Group 2 (diode laser) the average dentinal tubular diameter to be 2.28 microns. Mean diameter of dentinal tubules in Group 3 (nanoHA alone) was 2.41 microns. The specimens belonging to Group 4 (DL + nanoHA) showed average dentinal tubular diameter were 1.74 microns. Conclusion The single application of nanocrystalline hydroxyapatite powder and diode laser irradiation, showed significantly greater tubular occlusion and increased reduction in dentinal tubular diameters showing greater potential in occluding open dentinal tubules when compared to diode laser alone.