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Abstract The rational synthesis of alternative materials is highly demanding due to the outbreak of infectious diseases and resistance to antibiotics. Herein, we report a tailored nanoantibiotic synthesis protocol where the antibiotic binding was optimized on the silver-silica core-shell nanoparticles surface to maximize biological responses. The obtained silver nanoparticles coated with mesoporous silica functionalized with ampicillin presented remarkable antimicrobial effects against susceptible and antibiotic-resistant Escherichia coli. In addition, these structures were not cell-death inducers and different steps of the mitotic cell cycle (prophase, anaphase and metaphase) were clearly identified. The superior biological results were attributed to a proper and tailored synthesis strategy.