A highly efficient oligomer antimicrobial agent, polyhexamethylene guanidine hydrochloride (PHMGH), is added to mixtures of polyvinylpyrrolidone (PVP)/chitosan (CS) solution to form antimicrobial PVP/CS/PHMGH (PCP) nanofibrous membranes using an electrospinning method. The solution parameters including viscosity and electrical conductivity are used to characterize the formation of the nanofibers, and then the antimicrobial activity, biocompatibility, and cell activity of the resultant nanofibrous membranes are evaluated. The results show that with the addition of 0.1-0.2% PHMGH, the nanofiber diameter reduces by 5-12%. The antimicrobial activity result shows that the addition of 0.16% PHMGH in PVP/CS (PCP-1) decreases the minimum inhibitory concentration against Escherichia coli to 138.1 mu g mL(-1), a factor of 2-4 times lower than the PVP/CS (PC) nanofibrous membrane. The in vitro result shows that PCP nanofibrous membranes still have good cell compatibility. These electrospun PCP nanofibrous membranes can become excellent candidates to be used as antimicrobial wound dressings in the future.