The proanthocyanidin (PA)-rich grape seed extract (GSE) is a collagen cross-linking agent that can perform a chemical bond with the dentin's collagen. The objective of this study was to evaluate the influence on shear bond strength (SBS) of the pre-conditioning of GSE, on human dentin surfaces conditioned with Er,Cr:YSGG laser. The sample consisted of 64 non-carious human teeth, divided into eight groups, four groups conditioned with Er,Cr:YSGG laser (4.5 W, 50 Hz, 50 μs, 70% air, 90% water) and four prepared with conventional methods (control). In both groups, a GSE solution was applied before using the two adhesives tested: Clearfil SE Bond (CSE) and Scotchbond Universal (SU). Subsequently, a SBS test, a scanning electron microscopy, and a statistical analysis were performed. In the laser groups, the best SBS mean (20.08 ± 4.01 MPa) was achieved in the group treated with GSE and CSE. The control group with the application of CSE showed the highest SBS mean (24.27 ± 10.28 MPa), and the group treated with laser and SU showed the lowest SBS mean (12.94 ± 6.51 MPa). Between these two groups there was a statistically significant difference (p = 0.05). However, this was not observed among the laser or control groups. The type of dentin surface preparation can influence the SBS. The CSE showed better SBS in laser and control groups. The presence of GSE did not improve the adhesion on surfaces conditioned with laser, but more studies should be carried out in the future to confirm this conclusion.