There are few therapeutic options to simultaneously tackle Staphylococcus aureus and Pseudomonas aeruginosa, two of the most relevant nosocomial and antibiotic-resistant pathogens responsible for implant, catheters and wound severe infections. The design and synthesis of polymers with inherent antimicrobial activity have gained increasing attention as a safe strategy to treat multi-drug-resistant microbes. Here, we tested the activity of a new. polymeric derivative with glycopolypeptide architecture (PAA-VC) bearing L-arginine, vancomycin, and colistin as side chains acting against multiple targets, which give rise to a broad spectrum antimicrobial activity favorably combining specific and nonspecific perturbation of the bacterial membrane. PAA-VC has been tested against planktonic and established biofilms of reference strains S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 and susceptible or antibiotic resistant clinical isolates of the above-mentioned microorganisms. MIC values observed for the conjugate (48-190 and 95-190 nM for P. aeruginosa and S. aureus strains, respectively) showed higher efficacy if compared with the free vancomycin (MICs within 1.07-4.28 mu M) and colistin (MICs within 0.63-1.33 mu M). Additionally, being highly biocompatible (IC50 > 1000, 430, and 250 mu g mL(-1) for PAA-VC, vancomycin and colistin respectively) high-dosage can be adopted for the eradication of infections in patients. This positively influences the anti-biofilm activity of the conjugate leading to a quasi-total eradication of established clinically relevant biofilms (inhibition >90% at 500 mu g mL(-1)). We believe that the in vitro presented data, especially the activity against established biofilms of two relevant pathogens, the high biocompatibility and the good mucoadhesion properties, would allow the use of PAA-VC as promising candidate to successfully address emerging infections.