To test the hypothesis that the expression profiles, evolutionary patterns, and functionalities of anti-microbial peptides (AMPs) and proteins in some hymenopteran and heteropteran species are influenced by their distinct ecological traits, we conducted venom and salivary gland-specific transcriptome analyses. We compared the expression and evolutionary patterns of anti-microbial peptide and proteins across 22 hymenopteran and 9 heteropteran species. Additionally, we investigated the structural properties of selected defensins and assessed their anti-microbial and hemolytic activities. Defensin expression in social wasps from the family Vespidae was higher than that from the family Apidae and in solitary hunting wasps. Among heteropterans, only 2 species C. lectularius and G. rubrolineatum showed higher transcript levels than any others examined. Notably, the social wasp V. flaviceps and the phytophagous heteropteran G. rubrolineatum exhibited the highest levels of defensin expression among the hymenopteran and heteropteran species studied, respectively. Most anti-microbial peptide and proteins, with the exception of peptidoglycan recognition protein-SC2 in hymenopteran species, exhibited evolutionary patterns that were distinct from the typical hymenopteran and heteropteran lineage structures. Among the defensins derived from hymenopteran and heteropteran species, as well as the traditional antibiotics tested, the defensin from the phytophagous heteropteran G. rubrolineatum exhibited the strongest Gram-positive bacteria-specific anti-microbial activity. These findings provide valuable insights into the evolutionary dynamics and functional diversity of hymenopteran and heteropteran anti-microbial peptide and proteins and may contribute to the development of novel AMP-based strategies as promising alternatives to conventional antibiotics.