SUMMARY Purpose This study aimed to evaluate the effects of two desensitizers and the Er,Cr:YSGG laser on human dentin tubules, applied alone or in combination. Methods Ninety-six dentin specimens were obtained from extracted third molars and divided into six groups: Group 1: no-treatment (Control); Group 2: nano-hydroxyapatite desensitizer (NhapD); Group 3: NhapD+Er,Cr:YSGG laser (L); Group 4: Er,Cr:YSGG laser (L); Group 5: glutaraldehyde desensitizer (GD); and Group 6: GD+L, respectively. All specimens were evaluated using scanning electron microscopy. The diameter and the number of open dentin tubules, the tubules’ occluding ratio, and the mineral coverage area were measured via the Image J software at 2000× magnification. Atomic force microscopy was used to determine the blocking mechanism of desensitizing treatments and the surface morphology of dentin specimens. One-way ANOVA and Tukey tests were used for statistical analysis. Results The number of open tubules and the mean diameter of tubules for all treatment groups showed statistically significant differences from the control group The NhapD+L group had a significantly lower number of open tubules than the L and GD groups. The NhapD+L and L groups significantly had higher occluding ratios than the other groups. Conclusions The present study showed that the Er,Cr:YSGG laser alone was effective in terms of tubule occlusion and also contributed to increasing the occluding ratio of nano-hydroxyapatite. It may be recommended to use the Er,Cr:YSGG laser with nano-hydroxyapatite desensitizers to achieve effective tubule occlusion.