The decline of insect pollinators is a significant concern within the current biodiversity crisis. The paradox between the benefits that these animals represent to humans and the evidence of human activities driving their extinction calls for the urgent protection of bees. To address the role of chemical pollution in this scenario, we assessed the acute toxicity as well as four biomarker responses (cholinesterase [ChE], glutathione S‐transferase, catalase, and lipid peroxidation [LPO]) elicited by dietary 24‐h exposure to three insecticides (malathion, imidacloprid, and fipronil) on the stingless neotropical bee Tetragonisca angustula and the honeybee Apis mellifera. Malathion was the most toxic substance to both species, with 48‐h median lethal doses (LD50s) of 0.25 ng/bee to A. mellifera and 0.02 ng/bee to T. angustula. Fipronil was also highly toxic and presented a similar toxicity to both species, with 48‐h LD50s of 0.5 ng/bee (A. mellifera) and 0.4 ng/bee (T. angustula). Imidacloprid had the lowest acute toxicity with a 48‐h LD50 of 29 ng/bee for A. mellifera, whereas T. angustula tolerated exposure higher than 35 ng/bee. Apparent biomarker responses were observed in bees of both species that survived exposure to higher concentrations of malathion (ChE inhibition) and fipronil (increased LPO). Our results suggest that specific sensitivity to insecticides varies greatly among compounds and pollinator species, but the use of different representative species can facilitate the prioritization of substances regarding their risk to pollinators. Further research is necessary to better characterize the risk that pesticides represent in neotropical agricultural landscapes. Environ Toxicol Chem 2023;42:1022–1031. © 2023 SETAC