<p>Quaternary ammonium salts (QASs) have emerged as a versatile class of compounds with significant potential across diverse biological applications due to their unique structural features, cationic nature, and broad-spectrum antimicrobial properties. They are effective against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and enveloped viruses. This review comprehensively explores the functionalization of QAS-based materials, highlighting their adaptability in designing advanced biocompatible systems. Special emphasis is placed on their roles in multifunctional antimicrobial agents with significant biological applications in infection control and healthcare materials such as antimicrobial coatings, drug delivery, gene transfection, and tissue engineering. Recent advances in tailoring QAS architectures to enhance selectivity, biostability, and efficacy are discussed, along with an overview of their mechanisms of action at the molecular level. The synthesis–structure–activity relationship elucidated herein provides critical insights for the future development of QAS materials with optimized biological performance. This article aims to serve as a valuable resource for researchers seeking innovative strategies to harness the full potential of quaternary ammonium salts in biomedical and pharmaceutical sciences.</p> Graphical abstract <p></p>