Silk fibroin protein, isolated from cocoons of the domesticated mulberry silkworm, Bombyx mori, finds extensive application in biomaterial design. In this study, poly(ethylene glycol) (PEG) 4000 has been used for blending fibroin from both B. mori and Antheraea mylitta, the wild tropical non-mulberry silkworm. PEG-blended films have shown marked changes from the pure fibroin films with respect to thermal properties and mechanical properties. FT-IR spectroscopy confirmed incorporation of new functional groups like quinone oximes. Pure fibroin and PEG-blended fibroin films showed biocompatibility with the HOS osteosarcoma cell line. Von Kossa staining confirmed nodule formation due to mineralization and differentiation of osteoblasts on pure and blended matrices. On account of increased surface roughness, higher elongation percentage, higher thermostability and better activity of osteoblasts in terms of intracellular alkaline phosphatase production, PEG-blended A. mylitta fibroin film shows better potential than PEG-blended B. mori fibroin film for use as potential biomaterial.