In this study, an asymmetric composite membrane composed of a porous chitosan layer and a dense polyhydroxybutyrate (PHB) layer was prepared, and its mechanical properties as well as cellular compatibility were evaluated. First, the PHB dense layer with a thickness of ca. 32 m was obtained by direct oven-drying. After being treated with plasma to produce free radicals on the surface and subsequently graft polymerized with poly(acrylic acid), the PHB was immersed in the CS solution where the CS chains could be chemically bonded to the PHB surface via carbodiimide activation. After lyophilization, an asymmetric PHB/CS composite membrane was obtained. Compared to the porous CS membrane, the composite membrane had higher tensile mechanical properties. In addition, the fibroblast cells (L929 and HGF) and human osteoblast cells (hFOB1.19) were cultured on both sides of the composite membrane. All the cells could proliferate very well indicating that both PHB and CS layers were cellular compatible. Yet, the PHB dense layer could prohibit the fibroblast cells to penetrate through the membrane. Thus, the asymmetric composite membrane can be use as a potential barrier membrane for guided bone regeneration in periodontal tissue engineering.