Purpose: To investigate the effect of erbium, chromium: yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser conditioning on dentin bond strength and nanoleakage of different universal and self-etch adhesives. Materials and Methods: A total of 84 intact human third molar teeth were cut at the dentin level, and half of them were laser conditioned. The specimens were divided into three groups; two different universal resins and one self-etch adhesive resin were applied and composite resin restorations were created. For the microtensile bond strength (µTBS) test, 20 microspecimens were prepared from the laser and control group of each adhesive (n = 20) and tested with a universal device. For nanoleakage observation, 10 specimens were prepared from each group (n = 10) and stored in silver nitrate solution, and then the amount of nanoleakage was analyzed by field emission scanning electron microscopy (FESEM). Data were analyzed with two-way ANOVA, Tukey’s honestly significant difference, and chi-square tests. Results: The mean dentin bond strength of all adhesives in the laser groups was found to be statistically significantly lower than those of the control groups (P < .05). No difference was found between the mean bond strength of the adhesives in the laser and control groups (P > .05). Higher nanoleakage was observed in all adhesives in the laser groups compared to the control groups (P < .05). Conclusions: Irradiation of the dentin surface with Er,Cr:YSGG could adversely affect the µTBS and nanoleakage, likely by affecting the structure of the hybrid layer.