The by-product of the slaughter house was utilized for the development of promising regenerative wound dressing material. Currently, dual-layered nanofibrous spongy scaffold was fabricated for tissue engineering applications. Herein, Keratin (K)-Fibrin (F)-Gelatin (G) 3D sponge loaded with Mupirocin (M) was fabricated with the naturally derived materials from bovine origin using freeze drying method. Moreover, poly(3-hydroxybutyric acid) (P) and Gelatin(G) solution loaded with Curcumin (C) were electrospun to get the dual drug loaded dual-layered nanofibrous spongy 3D scaffold (KFG:M-PG:C). The fabricated biomaterial was assisted with physical, biological and mechanical property. The in vitro cell viability and fluorescence staining of NIH 3T3 and HaCaT cells assist in cell adhesion and proliferation of the dual-layered scaffold. Moreover, in vivo assessment using silicone splint animal model was employed. The nanofibrous surface aids in the migration of fibroblast for the increased the collagen deposition and granulation tissue formation. Nonetheless, the 3D spongy surface promotes the gaseous exchange and absorption of exudates. The fabricated KFG:M-PG:C scaffold has the ability to produce perusable material that can integrate with the host tissue. Overall, the three dimensional (3D) dual-layered nanofibrous spongy scaffold with synergistic effect of dual drugs prevents from infection and facilitates as highly durable substrate in tissue engineering application. (C) 2017 Elsevier B.V. All rights reserved.