Infection remains a major challenge in wound care, and the alarming rise of antimicrobial resistance (AMR) has intensified the need for effective wound healing strategies. Recent technological advances have highlighted the potential of antimicrobial peptides (AMPs) as promising alternatives to conventional antibiotics in combating AMR. In this study, AMPs—specifically nisin and cathelicidin LL-37—were coated onto a commercially available dermal substitute, the biodegradable temporising matrix (BTM®), to develop a next-generation antimicrobial dermal substitute aimed at addressing infection in wound care. In vitro results demonstrated strong antimicrobial activity of the AMP-coated BTM and ability to eradicate mature biofilms of common Gram-positive and Gram-negative pathogens. AMP-coated BTM showed a comparable efficacy to the commercial antimicrobial matrix, Endoform® in a direct contact kill assay. Using ex vivo bioluminescent biofilm models, the AMP-coated BTM reduced the burden of metabolically active bacteria by >50%. Furthermore, a pilot study using an infected wound biofilm mouse model indicated the safety of the developed dermal substitute for use in vivo. Overall, tethering AMPs to dermal substitutes presents a promising approach for managing clinical infections and eradicating biofilms in chronic wounds.