Background: Tachyplesin III, an antimicrobial peptide (AMP), provides protection against multidrug-resistant (MDR) bacterial infections and shows cytotoxicity to mammalian cells. Mixed bacterial infections, of which P aeruginosa plus A. baumannii is the most common and dangerous combination, are critical contributors to the morbidity and mortality of long-term in-hospital respiratory medicine patients. Therefore, the development of effective therapeutic approaches to mixed bacterial infections is urgently needed. Methods and results: In this study, we demonstrated that compared with individual infections, mixed infections with MDR bacteria P aeruginosa and A. baumannii cause more serious diseases, with increased pro-inflammatory cytokines (IL-1 beta, IL-6, TNF-alpha) and chemokines (MCP-1/MIP-2) and reduced mouse survival. In vitro treatment with Tachyplesin III enhanced phagocytosis in a mouse alveolar macrophage cell line (MH-S). Strikingly, in vivo, Tachyplesin III demonstrated a potential role against mixed-MDR bacterial coinfection. The bacterial burden in bronchoalveolar lavage fluid (BALF) was significantly reduced in the Tachyplesin /H-treated group. In addition, a systemic reduction in pro-inflammatory cytokines and decreased lung injury occurred with Tachyplesin III therapy. Conclusion: Therefore, our study demonstrated that Tachyplesin III represents a potential therapeutic treatment against mixed-MDR bacterial infection in vivo, which sheds light on the development of therapeutic strategies against mixed-MDR bacterial infections.