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The nonspecific adsorption of proteins and bacteria on the surface of polydimethylsiloxane (PDMS) had been a serious concern in a wide range of applications, such as medical devices. In order to improve the anti-adhesive and antibacterial capability, bare silver nanoparticles (AgNPs, ~15 nm) were generated in-situ on their surface without extra reducing and stabilizing agents. The main reason for this was that the SiO2 microspheres that are covalent bonded to the bulked PDMS could not only generate AgNPs spontaneously but also insure that no AgNPs were released to the environment. Meanwhile, the thiol-group-functionalized SiO2 microspheres self-assembled on the surface of PDMS by thiol-vinyl click reaction without any impact on their biomedical applications. After the modification of SiO2 microspheres with AgNPs, the surface of PDMS showed a smaller water contact angle than before, and the adhesion and growth of E. coli and Bacillus subtilis were effectively inhibited. When the monolayer of SiO2 microspheres with AgNPs was assembled completely on the surface of PDMS, they present improved bacterial resistance performance (living bacteria, 0%). This approach offers an antibacterial and anti-adhesive surface bearing small and well-defined quantities of in-situ generated AgNPs, and it is a novel, green, simple, and low-cost technique to generate AgNPs on soft biomedical substrates.