Despite the fact that polystyrene (PS) spheres have been developed as polymeric carriers or matrices for various biomedical applications, the synthesis of PS spheres is time-consuming. This work describes the fabrication of a uniform PS sphere, coated with silver nanoparticles (Ag-PS), by simultaneous photoinduced polymerization and reduction fabricated using x-rays in aqueous solution without any initiator. The solution contains only styrene, silver ions (Ag(+)), and poly(vinyl pyrrolidone) (PVP) as a stabilizer. The proposed mechanism of the formation of the Ag-PS nanocomposite spheres involves the generation of radicals in the aqueous solution to induce PS polymerization and the reduction of Ag. The distribution of the sizes of the core PS spheres in the Ag-PS nanocomposite spheres was systematically examined as a function of irradiation time, concentration of styrene, and amount of PVP. Ag-PS nanocomposite spheres exhibit antimicrobial activity against bacteria (Escherichia coli and Staphylococcus aureus). Additionally, the cationic (vinylbenzyl)trimethylammonium (TMA) monomer was photopolymerized to form positively charged TMA-PS spheres as gene carriers with uniquely low cytotoxicity. Given these design advantages, the method proposed herein is simpler than typical approaches for synthesizing PS spheres with functionalized groups and PS spheres coated with Ag nanoparticles.