The opportunistic pathogen Pseudomonas aeruginosa is a significant contributor to recalcitrant multi-drug resistant infections. In a vigorous search for alternative therapeutic approaches, the communication system used by this bacterium to synchronise the expression of genes involved in pathogenicity has been identified as a potential target. Poly(ε-lysine) dendrons, composed of three branching generations, were examined herein for their anti-virulence potential and ability to disperse within P. a eruginosa biofilms. These hyperbranched macromolecules reduced attachment and biomass production under different nutrient growth conditions, and at concentrations that were not lethal to planktonic cells (0.2, 0.4 and 0.8 mg/mL). Fluorescent labelling revealed the intracellular localisation and cell-penetrating capacity of the dendron, and showed the rapid uptake and release of unexploited dendron from pre-established P. a eruginosa biofilms. Additionally, the dendron induced complete attenuation of LasA protease, a marker of quorum sensing inactivation, by preventing its accumulation in the external environment. This study thus demonstrates the anti-virulence potential of this class of macromolecules, and could represent a novel therapeutic approach for the treatment of antibiotic-resistant P. a eruginosa infections.