Development of new generation bionanotextiles is an important growing field, and they have found applications as wound dressings, bandages, tissue scaffolds, etc. In this study, silver nanoparticle (AgNP) containing silk-based bionanotextiles were fabricated by electrospinning, and processing parameters were optimized and discussed in detail. AgNPs were in situ synthesized within fibroin nanofibers by UV reduction of silver ions to metallic silver. The influence of post-treatments via methanol treatment and glutaraldehyde (GA) vapor exhibited changes in the secondary structure of silk. Methanol treatment increased the tensile properties of fibers due to supported crystalline silk structure, while GA vapor promoted amorphous secondary structure. AgNP containing silk fibroin bionanotextiles had strong antibacterial activity against gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa.