The aim of this clinical study was to determine and compare the efficiency of the glutaraldehyde-containing agent (GCA), Nd:YAG, Er,Cr:YSGG lasers, and the combination of them on the dentin hypersensitivity (DH) treatment.This study was performed with the participation of 17 healthy adult patients having 100 teeth with DH; the patients were randomly divided into five groups according to the treatment protocol: (1) application of GCA on sensitive teeth, (2) Nd:YAG laser (1 W/cm2, 10 Hz) irradiation on sensitive teeth, (3) application of GCA on sensitive teeth and then Nd:YAG laser irradiation, (4) Er,Cr:YSGG laser (0.25 W/cm2, 20 Hz) irradiation on sensitive teeth, (5) application of GCA on sensitive teeth and then Er,Cr:YSGG laser irradiation. Sensitivity levels were assessed by the Yeaple probe on the buccal surfaces of the teeth at a force setting of 10 g. Measurements were performed for 30 min, after 7, 90, and 180 days of the therapy to assess the effects of desensitization. The evaluations were analyzed using the one-way analysis of variance and repeated measurement test (P < 0.05).After sessions, DH was significantly reduced in all groups at each measurement point. The Er,Cr:YSGG laser with or without GCA application were the most effective ones in DH treatment (P < 0.05). Comparison of the treatment regimens demonstrated that the scores achieved with the Yeaple probe were not significantly higher for the Nd:YAG laser groups than the GCA alone group.This clinical study shows that the Er,Cr:YSGG laser have promising potential for the treatment of DH.