To comprehend the drivers underlying venom variation in ants, we selected 15 Neotropical species and recorded a range of traits, including ecology, morphology, and venom bioactivity. Principal component analysis of both morphological and venom bioactivity traits revealed that stinging ants display two functional strategies. Additionally, phylogenetic comparative analysis indicated that venom function (predatory, defensive, or both) and mandible morphology significantly correlate with venom bioactivity and amount, while pain-inducing activity trades off with insect paralysis. Further analysis of the venom biochemistry of the 15 species revealed switches between cytotoxic and neurotoxic venom compositions in some species. This study highlights the fact that ant venoms are not homogenous, and for some species, there are major shifts in venom composition associated with the diversification of venom ecological functions.