<p>Novel solutions to combat the rapid evolution of antimicrobial resistance in human and animal pathogens are urgently required. Antimicrobial peptides (AMPs) represent promising therapeutic molecules, as they exhibit structural nuances and distinct molecular targets against pathogenic microorganisms. In this Review, we explore the multifaceted structural nature of AMPs and advanced structural conformations, discuss the distinct mechanisms of action and explore novel targets. Additionally, we discuss resistance mechanisms, cross-resistance and innovative strategies for AMP design and optimization. We argue that gaining insight into novel AMP structural arrangements, targets and design optimization is crucial for the development of innovative therapies that can be translated into clinical as well as broader applications.</p>