Cell compatibility is one of the prominent requirements for the fabrication of tissue engineering materials. Silk fibroin (SF) is an excellent material for biomedical applications and shows desirable properties such as good compatibility, minimal tissue reaction, and tailorable degradability. Poly[(R)-3-hydroxybutyrate] (PHB) has a high percentage of crystallinity and a high melting temperature. In this study, PHB and SF were blended together to improve the mechanical properties of the SF fibrous structure and the crystallinity of PHB. Furthermore, the cell-biomaterial interaction on the PHB/SF composite scaffold was expected to be enhanced via the SF. For this purpose, PHB/SF composite scaffolds were prepared through the use of the electrospinning technique, which is a unique method for the production of biomaterials on the nanoscale intended for tissue engineering. PHB/SF scaffolds were prepared with different SF contents and a suitable electrospinning condition was chosen in terms of structure and fiber diameter. The average fiber diameter was 92 ± 2.6 nm at a flow rate of 0.4 mL/h, distance of 20 cm, polymer concentration of PHB/SF-0 of 7%:18% (w/w) or 1:1 (v/v), and voltage of 20 kV. Mechanical and crystalline properties of the PHB/SF scaffold were investigated. Adhesion and proliferation of L929 and HaCaT (mouse fibroblast and immortalized human keratinocyte cell lines) on PHB/SF-0 were examined.