Nanomaterials as antimicrobial and antibiofilm agents: mechanistic insights and limitations of current testing principles.

Antimicrobial resistance (AMR) and recalcitrant biofilm-associated infections remain two of the most critical challenges in modern medicine, emphasizing the need for innovative therapeutic strategies. Against this backdrop, antimicrobial nanomaterials (ANMs) represent a promising class of therapeutic agents, offering unique opportunities for addressing infections where conventional antibiotics fail, including biofilm-associated and multidrug-resistant (MDR) infections. ANMs leverage unique nanoscale properties to exert their activity through multiple mechanisms of action, rather than through the delivery of conventional antibiotic payloads. Their multimodal activity has also been suggested to reduce selective pressure for resistance development. Despite their potential, the field is held back by a critical, largely unaddressed methodological bottleneck: the absence of robust, harmonized evaluation protocols specifically adapted to the particulate, dynamic, and multi-mechanistic nature of ANMs. Standard antimicrobial susceptibility assays, designed for soluble small-molecules, often produce systematic artefacts and interference when applied to particulate systems, ultimately compromising the reliability and comparability of reported data. This review provides a comprehensive analysis of the physicochemical determinants governing ANMs interactions with bacteria and biofilms, together with an overview of the major antimicrobial mechanisms against both planktonic and biofilm systems and the experimental approaches currently used to support mechanistic claims. By critically evaluating the limitations of existing regulatory frameworks and characterization workflows, this work aims to establish a reliable foundation for the reproducible evaluation and practical application of the next generation of ANMs.