Beauveria bassiana and Bacillus thuringiensis are widely utilized entomopathogens for biological control. Insects combat these microbial threats through the activation of innate immune defenses, with antimicrobial peptides (AMPs), particularly defensins, playing pivotal roles. Tuta (Phthorimaea) absoluta (Lepidoptera: Gelechiidae), is a rapidly spreading and highly destructive invasive tomato pest that has developed resistance to conventional insecticides. To enhance the efficacy of entomopathogens targeted by AMPs, we investigated defensin functions in T. absoluta during pathogen challenge. A genome-wide survey identified four defensin genes (tadef1–4), each encoding 76–124 amino acids (228–372bp ORFs). All contained the insect defensin motif (C-X5-16-C-X3-C-X9-10-C-X4-7-CX1-C) and a conserved two-exon structure. Phylogenetic analysis confirmed their membership in the insect defensin family. Post-infection expression profiling showed that tadef1/2 were upregulated by both B. bassiana and B. thuringiensis, while tadef3/4 were suppressed by B. bassiana but induced by B. thuringiensis relative to uninfected controls. Developmental and tissue-specific expression analyses showed that tadef1/2 peaked in early instar larvae, especially midgut. Recombinant tadef1/2 demonstrated antibacterial activity against B. thuringiensis but lacked antifungal effects against B. bassiana. These results establish tadef1/2 as key effectors against Gram-positive bacteria and provide a molecular basis for improving the biocontrol of T. absoluta via immune modulation.