Leaf-cutter ants are agricultural and urban pests that defy chemical control methods. Laboratory and field studies have revealed repellent and insecticidal activity by the extracts of Tithonia diversifolia (Asteraceae), known as Mexican sunflower, as a promising alternative for the control of the leaf-cutter ant Atta cephalotes. This study evaluated the effects of different extracts (non-polar and polar) of T. diversifolia dry leaves on worker ants from laboratory colonies of A. cephalotes through ingestion and contact. In addition, the biological activity of the extracts as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was evaluated. A dichloromethane extract at 1000 ppm presented the highest insecticidal activity through ingestion, causing 70% and 90% worker ant mortality after five and seven days of treatment, respectively. The acetylcholinesterase inhibition values showed that the dichloromethane presented the best AChE concentration of inhibition (IC50) at 73.9 ± 11.06 μg/mL, compared to its fractions, which demonstrates that its activity is potentiated when the crude extract is used. Our results can be attributed to the existence of terpenes and sesquiterpene lactones, which are likely inhibitors of AChE, in T. diversifolia.