Lugdunin was the first reported new class of thiazolidine cyclopeptide antibiotic from human microbiomes. However, its structure could not accommodate polar amino acids or modules, limiting its potential for chemical modification and clinical application. Herein, we developed a combinatorial modification strategy based on in situ modification of tryptophan through a radical reaction and amino acid site-specific mutation, transforming lugdunin into a cationic cyclic peptide antibiotic. Among the derivatives of lugdunin, WK6 was identified as a highly potent membrane-active antibiotic with rapid bactericidal activity and low resistance development potential. Remarkably, it showed therapeutic efficacy in MRSA-infected murine models of keratitis, pneumonia, and peritonitis. Additionally, when grafted onto contact lens Surfaces, WK6 exhibited potent antifouling capabilities, highlighting its potential in implant antifouling applications. Therefore, this study developed an effective strategy to optimize lugdunin and unveiled a novel cationic lugdunin derivative WK6, which could be recognized as a promising lead compound to combat multidrug-resistant bacteria.