In this study, the bi-layered disulfiram-loaded fiber membranes with antibacterial activity and different surface wettabilities are prepared using electrospinning technology. In the application of wound dressing, the hydrophilic surface of fiber membranes is beneficial for the cell adhesion and drug release to heal the wound, meanwhile the hydrophobic outside surface is able to block water penetration to reduce the probability of wound infection. The obtained bi-layered drug-loaded fiber membranes are composed of polyvinylidene fluoride (PVDF) bottom surface and disulfiram (DSF)/polylactic acid (PLA) top surface. To modify the top surface wettability, the oxygen plasma modification of bi-layered membranes was carried out. We analyzed the morphology, wettability and chemical compositions of bi-layered drug-loaded fiber membranes by various techniques. And the bi-layered disulfiram-loaded membranes showed potent antibacterial activity in vitro against both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Thus, the obtained bi-layered disulfiram-loaded fiber membranes are very suitable for wound dressings application.