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Background/Objectives: Bacterial biofilms formed by Escherichia coli pose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis. Methods: In vitro assays included MIC determination, biofilm eradication testing (crystal violet, colony counts, CLSM), swimming motility, and EPS quantification. CRISPR/Cas9 was used to construct and complement a kduD mutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In vivo, a murine excisional wound infection model was treated with CRAMP-34, with wound closure and bacterial burden monitored. Gene expression changes were analyzed via RT-qPCR. Results: The mouse-derived AMP (abbreviation CRAMP-34) effectively eradicates pre-formed biofilms of a clinically relevant, porcine-origin E.coli strain and promotes wound healing in a murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified kduD, as a critical gene for robust biofilm formation. Functional characterization revealed that kduD deletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of the kduD deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. Conclusions: The research highlight CRAMP-34 as a potent anti-biofilm agent and unveil kduD as a previously unrecognized regulator of E.coli biofilms development, whose associated pathway is implicated in the mechanism of action of CRAMP-34.