The effect of the solvents for silk fibroin (SF) extraction on its antimicrobial activity was studied. Extraction protocols were performed using LiBr (SF_L) and Ajisawa’s reagent (CaCl₂:ethanol:H₂O) (SF_C). The morphological and structural characteristics of the extracted SF and their composites were assessed. Corresponding bactericidal activities against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Pseudomonas aeroginosa (ATCC 27853) were performed. The resulting solutions were either casted into films or individually incorporated into composites of silver nanoparticles (NS) embedded into chitosan fragments (Cs) through γ-irradiation. Films of SF, obtained by using the two solvents, as well as the final prepared composites of SF, NS and Cs were analyzed using XRD, FTIR, SEM, TEM and zeta potential at several pH values. The band gap values were calculated. The results proved that, although SF_C consumed shorter gelation time, yet SF_L exerted higher antibiotic activity against the tested microorganisms. Moreover, the final composites had the ability to significantly reduce the growth of these medically relevant bacteria and are, therefore, recommended as a novel natural antibacterial biomaterial for several biomedical applications.