In this study, we isolated and purified cyclic lipopeptide PDMA from our in-house strain Pseudomonas tolaasii ZTB4. We evaluated its antibacterial and antibiofilm activities against VREF, demonstrating that PDMA effectively eradicates mature biofilms. To decipher the mechanisms underlying this eradication, we employed an integrated multi-omics approach combining transcriptomics, proteomics, and metabolomics to identify key intracellular targets. This represents the first study to investigate the antibiofilm mechanism of PDMA using multi-omics analysis, along with hemolytic assay to evaluate biosafety for future structure-activity relationship analysis. Our findings highlight PDMA as a promising scaffold, underscoring its potential for structural optimization and application in combating biofilm-related drug tolerance.