This study evaluated the effects of 10% carbamide peroxide, carbopol and glycerin and their associations on microhardness over time on enamel and dentin. Eight treatment agents were evaluated: a commercial bleaching agent containing 10% carbamide peroxide (Opalescence 10% Ultradent), 10% carbamide peroxide, carbopol, glycerin, 10% carbamide peroxide + carbopol, 10% carbamide peroxide + glycerin, carbopol + glycerin and 10% carbamide peroxide + carbopol + glycerin. Three hundred and twenty human dental fragments, 80 sound enamel fragments (SE), 80 demineralized enamel fragments (DE), 80 sound dentin fragments (SD) and 80 demineralized dentin (DD) fragments, were exposed to the treatment agents (n=10). These agents were applied onto the surface of the fragments eight hours a day for 42 days. After eight hours, they were washed from the dental fragment surfaces after five back-and-forth movements with a soft bristle toothbrush under distilled and deionized running water. During the remaining time (16 hours per day), the fragments were kept in individual vials in artificial saliva. After the 42-day treatment period, the specimens were kept individually in artificial saliva for 14 days. Knoop microhardness measurements were performed at baseline, after eight hours, and 7, 14, 21, 28, 35 and 42 days, and 7 and 14 days post-treatment (corresponding to 49 and 56 days after the initial treatment agent applications). The non-parametric Kruskal-Wallis analysis showed significant differences among the agents at each time interval, except at baseline for sound and demineralized enamel and dentin. For SE, SD and DD, there was a decrease in microhardness values during treatment with all agents. There was a tendency towards lower microhardness values after treatment with carbopol and its associations for sound tissues. DD showed low microhardness values during and after treatment with CP and its associations. For DE, there was an increase in microhardness values during treatment with all agents and in the post-treatment phase. The baseline microhardness values were not recovered during the 14-day post-treatment phase. Opalescence 10%, carbamide peroxide, carbopol, glycerin and their associations may change the microhardness of sound and demineralized dental tissues, even in the presence of artificial saliva.