An electrospinning method was used to fabricate a bilayer collagen sponge/chitosan (Col/CS) matrix to develop a new wound healing dressing. The aim of the study was to improve the mechanical properties and antimicrobial activity of a collagen sponge matrix used as medical device. Col/CS matrix was subjected to detailed analysis by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Atomic force microscopy (AFM), comparative physico-chemical characteristics (swelling ratio and hydrolysis properties, enzymolytic properties, bilayer Col/CS morphology) between collagen sponge and Col/CS matrix, bioburden determination and antimicrobial activity. The physical characteristics, morphology and antimicrobial properties showed that this biomaterial has a high ratio of surface area and superior mechanical properties, than collagen sponge. After sterilization, the recovery efficiency of S. aureus and E. coli from Col/CS matrix was 36% and 52%, result confirmed through qualitative antimicrobial activity. These results suggest that Col/CS matrix could be a promising solution for chronic wounds management.