Introduction: Methods: The antimicrobial and anti-virulence activity of the iminosugar Results and discussion: L-NPDNJ exhibited weak bactericidal activity but showed additive effects when used in combination with amikacin and tobramycin, reducing their minimum inhibitory concentrations by two- to threefold. At sub-inhibitory concentrations, L-NPDNJ was found to significantly modulate the expression of genes involved in antibiotic resistance, biofilm regulation, and virulence, including those encoding efflux pumps, global regulators, and extracellular proteases. These transcriptional changes were found to be associated with significant phenotypic effects. At sub-inhibitory concentrations, L-NPDNJ reduced cell surface hydrophobicity by up to 90%, impaired adhesion to abiotic surfaces and to human lung epithelial cells by over 90%, and disrupted preformed biofilms, decreasing biofilm biomass and metabolic activity by approximately 70%. In addition, L-NPDNJ has been shown to enhance macrophage phagocytosis and reduce intracellular bacterial survival, while improving epithelial cell viability. Notably, pretreatment of Conclusion: Collectively, these findings demonstrate that L-NPDNJ exerts a broad anti-virulence effect against