Bionanocomposite packaging materials have a bright future for a broad range of applications in the food and biomedical industries. Antimicrobial packaging is one of the bionanocomposite packaging materials. Silver nanoparticle (AgNP) is one of the most attractive antimicrobial agents for its broad spectrum of antimicrobial activity against microorganisms. However, the traditional method of preparing AgNPs-functionalized packaging material is cumbersome and not environmentally friendly. To develop an efficient and convenient biosynthesis method to prepare AgNPs-modified bionanocomposite material for packaging applications, we synthesized AgNPs in situ in a silk fibroin solution via the reduction of Ag+ by the tyrosine residue of fibroin, and then prepared AgNPs–silk/poly(vinyl alcohol) (PVA) composite film by blending with PVA. AgNPs were synthesized evenly on the surface or embedded in the interior of silk/PVA film. The prepared AgNPs–silk/PVA film exhibited excellent mechanical performance and stability, as well as good antibacterial activity against both Gram-negative and Gram-positive bacteria. AgNPs–silk/PVA film offers more choices to be potentially applied in the active packaging field.