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Multidrug resistant bacterial infections threaten to become the number one cause of death by the year 2050. Development of antimicrobial dendritic polymers is considered promising as an alternative infection control strategy. For antimicrobial dendritic polymers to effectively kill bacteria residing in infectious biofilms, they have to penetrate and accumulate deep into biofilms. Biofilms are often recalcitrant to antimicrobial penetration and accumulation. Therefore, this work aims to determine the role of compact dendrons with different peripheral composition in their penetration into Pseudomonas aeruginosa biofilms. Red fluorescently labeled dendrons with pH-responsive NH 3 + peripheral groups initially penetrated faster from a buffer suspension at pH 7.0 into the acidic environment of P. aeruginosa biofilms than dendrons with OH or COO - groups at their periphery. In addition, dendrons with NH 3 + peripheral groups accumulated near the top of the biofilm due to electrostatic double-layer attraction with negatively charged biofilm components. However, accumulation of dendrons with OH and COO - peripheral groups was more evenly distributed across the depth of the biofilms than NH 3 + composed dendrons and exceeded accumulation of NH 3 + composed dendrons after 10 min of exposure. Unlike dendrons with NH 3 + groups at their periphery, dendrons with OH or COO - peripheral groups, lacking strong electrostatic double-layer attraction with biofilm components, were largely washed-out during exposure to PBS without dendrons. Thus, penetration and accumulation of dendrons into biofilms is controlled by their peripheral composition through electrostatic double-layer interactions, which is an important finding for the further development of new antimicrobial or antimicrobial-carrying dendritic polymers.