OBJECTIVES: The aim of this in vitro study was to evaluate the effect of incorporating three different nanobiomaterials into bleaching material on microhardness of bleached enamel. MATERIALS AND METHODS: The crowns of 24 extracted sound human molars were sectioned. Sixty enamel specimens (2 × 3 × 4 mm) were selected and divided into five groups (n = 12): Group 1 received no bleaching procedure (control); Group 2 underwent bleaching with a 40% hydrogen peroxide (HP) gel; Groups 3, 4, and 5 were bleached with a 40% HP gel modified by incorporation of bioactive glass (BAG), amorphous calcium phosphate (ACP) and hydroxyapatite (HA), respectively. The enamel microhardness was evaluated. The differences in Knoop microhardness data of each group were analyzed by one-way ANOVA, followed by post hoc Tukey tests. RESULTS: Significant differences were observed between the study groups. The enamel microhardness changes in Groups 1, 3, 4, and 5 were significantly lower than that of Group 2 (p < 0.001). CONCLUSIONS: Within the limitations of this study, it can be concluded that incorporation of each one of the three tested biomaterials as remineralizing agents might be effective in decreasing enamel microhardness changes subsequent to in-office bleaching.