Glyphosate stands out in the eucalyptus management, which makes it essential to know its behavior, its effects on the plant, and possible environmental impacts. This study aimed to identify and quantify the root exudation of glyphosate and aminomethylphosphonic acid (AMPA) by Eucalyptus urophylla with chromatographic and biological methods. The five glyphosate doses were tested (0, 360, 720, 1080 and 1440 g a.e ha−1) on E. urophylla plants. The physiological and intoxication evaluations were performed after herbicide application. Water samples remaining from the pots were used for chemical quantification of root exudation of glyphosate and AMPA in high-performance liquid chromatography. Cucurbita pepo plants were used as bioindicators of glyphosate in the water remaining in the pots after applying herbicide. The increase in glyphosate doses promoted linear growth in E. urophylla intoxication and significantly reduced total dry mass and root production. E. urophylla plants had their photosynthetic, transpiratory, and stomatal conductance rates reduced as the herbicide doses increased. The AMPA root exudation was not detected, but it was possible to identify the presence of glyphosate by bioassay and chemical methods. Root exudation of glyphosate by eucalyptus can result in lesser herbicide action in plant control and cause contamination of deeper soil layers.