Healthcare-associated infections (HAIs) remain difficult to manage due to the emergence of antibiotic-resistant pathogens and the persistence of biofilm-based infections. Thus, novel therapeutic strategies beyond conventional antibiotics are needed. In this study, we designed a multifunctional antimicrobial peptide, TR‑MOD, based on venom gland–specific transcriptomic data from the spider Araneus ventricosus . By introducing tryptophan and arginine substitutions, we increased the peptide’s hydrophobicity, net positive charge, and generated a pronounced amphipathic structure to enhance interactions with the bacterial membrane. TR‑MOD exhibited antibacterial activity against Pseudomonas aeruginosa and effectively inhibited biofilm formation, even at sub-minimum inhibitory concentrations. Mechanistic study revealed that TR-MOD disrupted bacterial morphology, induced extracellular polymeric substance redistribution, and modulated c-di-GMP–associated gene expression, indicating a multitarget mode of action. Since P . aeruginosa biofilms directly contribute to impaired wound healing via sustained inflammation and extracellular matrix disruption, we evaluated whether TR‑MOD could promote host tissue repair. TR‑MOD promoted fibroblast migration via activation of the phosphoinositide 3-kinase/AKT signaling pathway, which was partially regulated by receptor tyrosine kinase signaling. Collectively, these findings demonstrate that TR-MOD is a multifunctional peptide capable of simultaneously controlling infection and promoting fibroblast migration, providing a basis for its further preclinical evaluation in the context of biofilm-associated HAIs.