When glyphosate is applied, some of the herbicide can reach adjacent vegetation and impact the natural plant community structure and diversity over the long term. This study evaluated the response of leaves of Zeyheria tuberculosa (Vell.) Bureau ex Verl. (Bignoniaceae) to the herbicide glyphosate. Plants were exposed to aerial applications of the herbicide at concentrations of 0, 360, 720, 1080, or 1440 g acid equivalent (a.e.)·ha−1. The concentrations of shikimic acid in leaves of the herbicide-treated plants were always higher than the controls. Visual symptoms became apparent four days after the application of 720 g·ha−1. Glyphosate induced an increase in malondialdehyde in Z. tuberculosa leaves. The lowest levels of chlorophyll a were found for the three last applied doses, and protein content decreased with the glyphosate treatment. Necrosis was observed on the epidermis and in the mesophyll. Glandular trichomes were also plasmolyzed. On the midrib there was plasmolysis of non-lignified cells. Micromorphologically, there were cell plasmolysis and rupture of glandular trichomes. Thus, glyphosate is phytotoxic to Z. tuberculosa by causing biochemical, anatomical, and morphological changes. The morphoanatomical injuries found on Z. tuberculosa were severe, suggesting that the presence of glyphosate can irreversibly affect this species and compromise its survival.