Biofilms in chronic wounds, including diabetic foot ulcers, pressure ulcers, and venous leg ulcers, pose a major challenge to wound management. Herein, we report a Janus-type antimicrobial dressing for eradication of biofilms in chronic wounds. The dressing consists of electrospun nanofiber membranes coupled with dissolvable microneedle arrays to enable effective delivery of a database-designed antimicrobial peptide to both inside and outside biofilms. This antimicrobial dressing exhibited high efficacy against a broad spectrum of resistant pathogens in vitro. Importantly, such a dressing was able to eradicate methicillin resistant Staphylococcus aureus (MRSA) biofilms in both an ex vivo human skin wound infection model and a type II diabetic mouse wound infection model after daily treatment without applying surgical debridement. Most importantly, the dressing can also completely remove the Pseudomonas aeruginosa and MRSA, dual-species biofilm in an ex vivo human skin infection model. In addition, our computational simulations also suggested that microneedles were more effective in the delivery of peptides to the biofilms than free drugs. Our results indicate that the Janus-type antimicrobial dressings may provide an effective treatment and management of chronic wound polymicrobial infections.