As an effective natural antibacterial component, the low water solubility of thymol (THY) has stemmed its potential in biomedical application. Here, beta-cyclodextrin (beta-CD) and THY were self-assembled to form water-soluble inclusion complex (IC). The successful formation of IC was confirmed via H-1 NMR. As an antibacterial agent, the resultant IC was then incorporated into cellulose acetate (CA) fibrous matrix with hierarchical structure (nanopores on porous fibrous webs) via electrospinning (CA/THY/beta-CD), and the pure THY was also encapsulated into CA for comparison (CA/THY). In vitro dissolution tests demonstrated that CA/THY/beta-CD fibrous membrane exhibited sustained drug release, which abided by non-Fickian diffusion. Besides, the CA/THY/beta-CD fibrous membrane exhibited more effective and long-lasting antibacterial activity against S. aureus. Furthermore, the combination of hierarchical porous structure with sustained drug release endowed the CA/THY/beta-CD fibrous membrane with good cytocompatibility. Taken together, the CA/THY/beta-CD fibrous membrane could be an attractive candidate for wound dressing material.