The need exists for biomaterials that prevent biofilm formation and associated infections. In this report, we have studied the synthesis, processing, and antimicrobial behavior of new silver-containing thermoplastic hydrogel nanofibrous webs. Thermoplastic hydrogels were synthesized from multiblock PEG-POSS polyurethanes (PEG: poly(ethylene gylcol); POSS: polyhedral oligosilsesquioxane) and electrospun into nanofibrous webs (diameter similar to 150 nm), with or without AgNO3. The nanofibrous hydrogels exhibited unusual shrinkage during water uptake, yielding a uniquely dense structure compared to hydrogels prepared from east films. Antimicrobial activity was examined using exposure to Escherichia coli with daily refreshment of medium and inoculation to a controlled cell density. Nanofibrous hydrogels without silver featured the most rapid and most extensive biofilm formation, while the silver-containing nanofibrous hydrogel featured outstanding biofilm resistance, with biofilm formation taking hold only after 14 days of incubation in daily refreshed bacterial cultures. We envision application of the unique antimicrobial hydrogels as wound dressings that combine sustained bactericidal properties and lack of volumetric swelling during water uptake.