Infectious diseases caused by pathogenic bacteria, fungi, and viruses pose a global health threat, aggravated by antimicrobial resistance (AMR). This study explores the antimicrobial efficacy of two nanostructured composite coatings —aluminum-doped zinc oxide with silver (AZO-Ag) and titanium dioxide with silver (TiO2-Ag)— on polyester substrates, against high-priority pathogens: susceptible and resistant Klebsiella pneumoniae and Staphylococcus aureus bacterial strains, Candida albicans clinical fungal strain, and H1N1 influenza virus. A multimodal analytical approach, encompassing SEM, live/dead fluorescence assays, CFU counts, RT-qPCR, and immunofluorescence, supported by ROS quantification, is adopted to evaluate antimicrobial activity and contribution of indirect and direct-contact mechanisms. Both coatings exhibit robust antimicrobial effects especially through indirect mechanisms, with TiO2-Ag generally more effective than AZO-Ag through indirect interactions while AZO-Ag more effective in some direct-contact scenarios. Bacterial reduction up to ≈99% and viral inactivation of 98% are observed. Cytotoxicity assays reveal some decline in fibroblast viability, underscoring the coatings’ potential mainly for non-clinical applications, like packaging or high-touch surfaces. Moreover, the study emphasizes limitations in using single antimicrobial tests and necessity of combining diverse methodologies to comprehensively assess performance and mechanisms of action, finally proposing strategic guidelines for selecting appropriate antimicrobial materials and evaluation techniques tailored to specific application contexts. © 2025 Elsevier B.V., All rights reserved.

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