Introduction In recent decades, the antimicrobial surfaces/coating properties towards a longlasting microbicidal effect have drawn enormous attention by researchers, they have been developed and used in a wide variety of high-touch hospital devices as a potential approach. MethodsIn this work, Ag NPs was synthesized by sputtering method at the different annealing temperatures of 100◦C, 200◦C, 300◦C, and 400◦C. Results As a result, the as-synthesized Ag-300 exhibits the highest E. coli antibacterial performance compared with others. This can be attributed to thechange of the Ag NPs toxicity based on the growth of nanoparticles during the deposition process related to the Ostwald ripening process, thermal activation and coalescence particles. Conclusion This work provides an essential insight into the antimicrobial activity of Ag NPs-based films synthesized through the vacuum deposition technique, resulting in opening a new approach for enhancing the antimicrobial efficacy and prospects