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In the past two decades, the increase in microbial resistance to conventional antimicrobials has spurred scientists around the world to search tirelessly for new treatments. Synthetic amino acid-based surfactants constitute a promising alternative to conventional antimicrobial compounds. In this work, two new cationic amino acid-based surfactants were synthesized and their physicochemical, antifungal and antibiofilm properties evaluated. The surfactants were based on phenylalanine-arginine (LPAM) and tryptophan-arginine (LTAM) and prepared from renewable raw materials using a simple chemical procedure. The critical micelle concentrations of the new surfactants were determined by conductivity and fluorescence. Micellization of LPAM and LTAM took place at 1.05 and 0.54 mM, respectively. Both exhibited good antifungal activity against fluconazole-resistant Candida spp. strains, with a low minimum inhibitory concentration (8.2 μg/mL). Their mechanism of action involves alterations in cell membrane permeability and mitochondrial damage, leading to death by apoptosis. Furthermore, when LPAM and LTAM were applied with Amphotericin B, a significant synergistic effect was observed against all the studied Candida strains. These new cationic surfactants are also able to disperse biofilms of Candida spp. at low concentrations. The results indicate that LPAM and LTAM have potential application to combat the advance of fungal resistance as well as microbial biofilms., (Copyright © 2021 Elsevier B.V. All rights reserved.)