Biomedical synthetic polymers have been used in soft and hard tissue regeneration because of their good processability and biodegradability. However, biomaterials such as poly(ε-caprolactone) (PCL) have various shortcomings, including intrinsic hydrophobicity and lack of bioactive functional groups. The material must be reinforced with natural biomaterials to achieve good cellular and mechanical performance as biomedical material. We fabricated a biocomposite using PCL and silk fibroin (SF) powder, which has good biocompatibility and mechanical properties. The hydrophilicity, mechanical properties and cellular behavior of the PCL/SF fibers were analyzed. In addition, we obtained a highly oriented conduit of electrospun biocomposite fibers by modifying the rolling collector of the electrospinning system. As the alignment of micro/nanofibers increased, the orthotropic mechanical properties were improved. The biocompatibility of the biocomposite was evaluated in a culture of bone-marrow-derived rat mesenchymal stem cells. The cellular result demonstrated the potential usefulness of electrospun biocomposites for various biomedical conduit systems.