Leaf-cutting ants are difficult pests to control because they have numerous defense strategies and are highly selective in their plant harvesting choices. The search for effective pest control methods that have minimal negative effects on the environment has been continuous. Azadirachtin, a compound extracted from the neem tree (Azadirachta indica), is a promising alternative for the control of various pests, as it is toxic to some insects but readily degrades in the environment. In this study, we evaluated the effects of azadirachtin on the mortality, through topical exposure to the compound, and immune response, by introducing an artificial antigen into leaf-cutting ants Atta sexdens and Acromyrmex subterraneus subterraneus. Azadirachtin caused death to minor and major workers of both species in a concentration-dependent manner. Topical application of the compound did not diminish the immune response of ants in a microfilament encapsulation assay. Azadirachtin showed no effect on the immune response of workers but increased worker mortality, which indicates its potential as an ant control agent.