Chronic infections represent a significant global health and economic challenge. Biofilms, which are bacterial communities encased in an extracellular polysaccharide matrix, contribute to approximately 80% of these infections. In particular, pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from the sputum of patients with cystic fibrosis and are commonly found in chronic wound infections. Within biofilms, bacteria demonstrate a remarkable increase in resistance and tolerance to antimicrobial treatment. We investigated the efficacy of combining the last-line antibiotic colistin with a membrane- and stringent stress response-targeting anti-biofilm peptide DJK-5 against co-biofilms comprised of multidrug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA). Colistin lacks canonical activity against S. aureus. However, our study revealed that under co-biofilm conditions, the antibiofilm peptide DJK-5 synergized with colistin against S. aureus. Similar enhancement was observed when daptomycin, a cyclic lipopeptide against Gram-positive bacteria, was combined with DJK-5, resulting in increased activity against P. aeruginosa. The combinatorial treatment induced morphological changes in both P. aeruginosa and S. aureus cell shape and size within co-biofilms. Importantly, our findings also demonstrate synergistic activity against both P. aeruginosa and S. aureus in a murine subcutaneous biofilm-like abscess model. In conclusion, combinatorial treatments with colistin or daptomycin and the anti-biofilm peptide DJK-5 show significant potential for targeting co-biofilm infections. These findings offer promising avenues for developing new therapeutic approaches to combat complex chronic infections.