A simple method to fabricate well-defined silver nanodots of different sizes using self-assembled polystyrene-b-poly(ethylene oxide) (PS-b-PEO) block copolymer was developed. The most well-defined nanodot patterns were observed using optimal concentrations of silver precursor (0.4, 0.6, and 1.2%) with average sizes of 10, 18, and 28 nm by different molecular weights of PS-b-PEO. Silver nanodot patterns were not observed at higher Ag precursor concentrations. In addition, after repeated depositions, the antimicrobial activity (AA) towards bacteria increased compared to well-defined nanodot arrangements. The AA of the silver nanodots was significantly affected by the concentration used independent of the particle size of the silver nanodots. Potentially, silver nanodots can be used as antimicrobial packaging application to preserve the quality of food products due to the data generated here demonstrated that these materials significantly delayed the growth of Pseudomonas fluorescens and Staphylococcus aureus.