Poly (vinyl pyrrolidone) (PVP)/cellulose nanocrystal (CNC)/silver nanoparticle composite fibers were prepared via electrospinning using N,N'-dimethylformamide (DMF) as a solvent. Rheology, morphology, thermal properties, mechanical properties, and antimicrobial activity of nanocomposites were characterized as a function of material composition. The PVP/CNC/Ag electrospun suspensions exhibited higher conductivity and better rheological properties compared with those of the pure PVP solution. The average diameter of the PVP electrospun fibers decreased with the increase in the amount of CNCs and Ag nanoparticles. Thermal stability of electrospun composite fibers was decreased with the addition of CNCs. The CNCs help increase the composite tensile strength, while the elongation at break decreased. The composite fibers included Ag nanoparticles showed improved antimicrobial activity against both the Gram-negative bacterium Escherichia coli (E. coli) and the Gram-positive bacterium Staphylococcus aureus (S. aureus). The enhanced strength and antimicrobial performances of PVP/CNC/Ag electrospun composite fibers make the mat material an attractive candidate for application in the biomedical field.