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Difficult-to-treat bacterial infections caused by resistant human and plant pathogens severely afflict hospitals, and concern the agri-food sectors. Bacteria from the Pseudomonadaceae family, such as <i>P. aeruginosa, P. putida, P. fluorescens,</i> and <i>P. straminea,</i> can be responsible for severe nosocomial infections in humans. <i>P. fragi</i> is the major cause of dairy and meat spoilage, while <i>P. syringae</i> can infect a wide range of economically important plant species, including tobacco, kiwi, and tomato. Therefore, a cationic water-soluble lysine dendrimer (G5-PDK) was tested on several species of <i>Pseudomonas</i> genus. Interestingly, G5-PDK demonstrated variable minimum inhibitory concentrations (MICs), depending on their pigment production, on <i>Pseudomonas aeruginosa</i> (1.6-> 6.4 µM), MICs = 3.2–6.4 µM on <i>P. putida</i> clinical isolates producing pyoverdine, and very low MICs (0.2–1.6 µM) on strains that produced non-pigmented colonies. Time-kill experiments established the rapid bactericidal activity of G5-PDK. In the cytotoxicity experiments on human keratinocytes, after 4 h of treatment with G5-PDK at concentrations 16–500 × MIC, more than 80% of viable cells were observed, and after 24 h, the selectivity indices were maintained above the maximum value reported as acceptable. Due to its proven bactericidal potency and low cytotoxicity, G5-PDK should be seriously considered to counteract clinically and environmentally relevant <i>Pseudomonas</i> isolates.