AbstractBacterial biofilms cause 65% of all human infections and are highly resistant to antibiotic therapy but lack specific treatments. To provide a human organoid model for studying host-microbe interplay and enabling screening for novel antibiofilm agents, a human epidermis organoid model with robust methicillin-resistant Staphylococcus aureus (MRSA) USA300 and Pseudomonas aeruginosa PAO1 biofilm was developed. Treatment of 1-day and 3-day MRSA and PAO1 biofilms with antibiofilm peptide DJK-5 significantly and substantially reduced the bacterial burden. This model enabled the screening of synthetic host defense peptides, revealing their superior antibiofilm activity against MRSA compared to the antibiotic mupirocin. The model was extended to evaluate thermally wounded skin infected with MRSA biofilms resulting in increased bacterial load, cytotoxicity, and pro-inflammatory cytokine levels that were all reduced upon treatment with DJK-5. Combination treatment of DJK-5 with an anti-inflammatory peptide, 1002, further reduced cytotoxicity and skin inflammation.