Antimicrobial peptides (AMPs) are one of the most diverse classes of antibiotics. A prominent example is polymyxin E (colistin), used as last-resort antibiotic against multidrug resistant (MDR) Gram-negative bacteria. However, the emergence of resistance and toxicity of colistin restrict its use. Dendrimer antimicrobial peptides are currently explored as an alternative to colistin, since they show low hemolytic activity and are active against MDR isolates of Pseudomonas aeruginosa and Acinetobacter baumannii. To unravel the mode of action and potential resistance mechanisms of dendrimer AMPs, spontaneous resistant mutants were selected on polymyxin B and the prototypical dendrimer G3KL in ESKAPE pathogens. We propose a model for decreased susceptibility to G3KL in P. aeruginosa that includes two modifications of the lipopolysaccharide resulting in decreased negative charges at the bacterial surface. We hypothesized that mechanisms leading to decreased susceptibility to G3KL may be different from those conferring polymyxin resistance and seem to be species-specific in Gram-negative bacteria.