The growing antibiotic resistance phenomenon continues to stimulate the search for new compounds and strategies to combat bacterial infections. In this study, we designed and synthesized a new polycationic macrocyclic compound (<b>2</b>) bearing four <i>N</i>-methyldiethanol ammonium groups clustered and circularly organized by a calix[4]arene scaffold. The <i>in vitro</i> activity of compound <b>2</b>, alone and in combination with known antibiotics (ofloxacin, chloramphenicol or tetracycline), was assessed against strains of <i>Staphylococcus aureus</i> (ATCC 6538 and methicillin-resistant isolate 15), <i>S. epidermidis</i> (ATCC 35984 and methicillin-resistant isolate 57), and <i>Pseudomonas aeruginosa</i> (ATCC 9027 and antibiotic-resistant isolate 1). Calix[4]arene derivative <b>2</b> showed significant antibacterial activity against ATCC and methicillin-resistant Gram positive Staphylococci, improved the stability of tetracycline in water, and in combination with antibiotics enhanced the antibiotic efficacy against Gram negative <i>P. aeruginosa</i> by an additive effect.