Lower tract respiratory infections caused by Gram-negative pathogens classified by the World Health Organization as priority targets (e.g., Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae) pose a global public health concern as they are often mediated by biofilms, including multidrug-resistant strains [1,2]. The rise of antimicrobial resistance in these complicated infections has revived the interest in polymyxins (PM) as a last resort treatment. However, despite PMs high efficacy, PM use has been reported to be limited by their nephro and neurotoxicity and emerging resistance [3]. So, there is an increasing interest in developing new PM derivatives and providing insight into their structure-activity relationships. The ultimate goal is to obtain novel PM-based structures with reduced toxicity and improved antimicrobial activity, including against biofilms (often neglected in PM design). Bearing this in mind, the two polymyxins - B (PMB) and E (PME) in clinical use were chosen as scaffolds for the synthesis of the novel derivatives by incorporation of an unnatural alpha,alpha-dialkylglycine at position 7 and by changing the N-terminal fatty acid of the native PM structure, using microwave-assisted solid-phase peptide synthesis. The synthesised derivatives were then evaluated for their antimicrobial activity against P. aeruginosa cultures, by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Their antibiofilm activity was also determined against 24 hold P. aeruginosa biofilms, and the number of biofilm cells estimated by CFU plating. Results were compared to those obtained for commercial PMB/PME.