Objectives: The aim of this study was to evaluate the efficacy of using erbium, chromium-doped:yttrium scandium gallium garnet (Er,Cr:YSGG) laser-treated dentine in a dentine barrier test device. Materials and Methods: The test materials (G-Bond™ and Vitrebond™) were applied onto laser-treated or laser-untreated dentine discs. After 24 h of exposure with perfusion of the test chamber, cell survival was evaluated based on enzyme activity and compared to a nontoxic control material. The mean of the control was set to 100% viability. Data were analyzed using the one-way analysis of variance and the Tukey's honest significant difference tests. Results: The responses of bovine pulp-derived cells after exposure to G-Bond and Vitrebond on Er,Cr:YSGG laser-treated and laser-untreated dentin were statistically different from negative control group (P < 0.05). Conclusion: Er,Cr:YSGG laser treatment was not successful enough in decreasing the cytotoxic effects of the dental materials. Different parameters of Er,Cr:YSGG laser or different laser types could be investigated as an alternative to minimizing the cytotoxic effects of dental materials.