The high incidence of antibiotic resistance and biofilm-associated infections is still a major cause of morbidity and mortality and triggers the need for new antimicrobial drugs and strategies. Nanotechnology is an emerging approach in the search for novel antimicrobial agents. The aim of this study was to investigate the inherent antibacterial effects of a self-assembling amphiphilic choline-calix[4]arene derivative (<b>Chol-Calix</b>) against Gram negative bacteria. <b>Chol-Calix</b> showed activity against <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i>, including antibiotic-resistant strains, and affected the bacterial biofilm and motility. The activity is likely related to the amphipathicity and cationic surface of <b>Chol-Calix</b> nanoassembly that can establish large contact interactions with the bacterial surface. <b>Chol-Calix</b> appears to be a promising candidate in the search for novel nanosized nonconventional antimicrobials.