Aim: To compare the effect after bleaching with 35% hydrogen peroxide with and without calcium gluconate on surface microroughness in an in vitro study on the bovine enamel. Materials and methods: The study had an in vitro experimental design. The following groups were formed: group I: MV (mesiovestibular) enamel, group II: V (vestibular) enamel, and group III: DV (dystovestibular) enamel, undergoing treatment with Whiteness HP Blue (H2O2 at 35% with calcium gluconate), Whiteness HP Maxx: H2O2 at 35% (without calcium gluconate), and physiological serum. A Surftest SJ-210 digital roughness meter (Mitutoyo) was used to evaluate microroughness. Results: The highest mean microroughness was found in group II (V) with 0.23 ± 0.13 µm and 0.17 ± 0.02 µm for Whiteness HP Blue and Whiteness HP Maxx, respectively. The post hoc analysis of surface microroughness subjected to H2O2 with and without calcium gluconate showed that there were only statistically significant differences between Whiteness HP Blue and the control (p = 0.032). Conclusion: There were no significant differences in surface microroughness on comparing the Whiteness HP Maxx group with the control group and the Whiteness HP Blue group. Clinical significance: The clinical importance of this study was that it allowed us to know the direct impact that bleaching agents with and without calcium have on dental structures.