Declining bee populations diminish pollination services, damaging plant, and agricultural biodiversity. One of the causes of this decline is the use of pesticides. Pesticides with glyphosate as an active ingredient are among the most used pesticides worldwide, being the most used in Brazil. This study determined the 24 and 48 hours LD50 (median lethal dose) of the herbicide glyphosate by ingestion, identified sublethal doses, and investigated its effects on the locomotion and behavior of Tetragonisca angustula workers. The LD50 found indicates that glyphosate is highly toxic to T. angustula. The doses applied, including concentrations found in nature and recommended dosage, caused death, motor changes (decreased speed and tremors), excessive self-cleaning, and disorientation (return to light and stop). These results suggest that this herbicide can negatively affect the pollination activity of T. angustula. Toxicity and sublethal effects evaluation of pesticides on bees contributes to a better understanding of the harmful effects on hives and to adopt strategies to reduce intoxication.