The aim of this study was to characterize antimicrobial electrospun poly(vinyl alcohol) (PVA) nanofibers containing benzyl triethylammonium chloride (BTEAC) as an antimicrobial agent. The antimicrobial BTEAC-PVA nanofibers were prepared through electrospinning at the optimal conditions of 15 kV voltage and a 1.0 mL h(-1) flow rate. Based on the minimum inhibitory concentration (MIC) test results against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and Klebsiella pneumonia, BTEAC-PVA nanofibers containing 2.6% BTEAC were fabricated to test the antibacterial and antiviral activities. The average diameter of the BTEAC-PVA nanofibers increased from 175.7 to 464.7 nm with increasing BTEAC concentration from 0 to 2.6%. The antimicrobial activities of the BTEAC-PVA nanofibers were tested against bacteria. The antibacterial tests with 2.6% BTEAC-PVA nanofibers demonstrated that bacterial reduction in PVA nanofibers was similar to the control value, indicating that PVA had a minimal effect on bacteria death. For the BTEAC-PVA nanofibers, the bacterial reduction ratio increased with increasing contact time, demonstrating that BTEAC-PVA nanofibers successfully inhibited the growth of bacteria. In addition, the antiviral tests against viruses (bacteriophages MS2 and PhiX174) showed that the BTEAC-PVA nanofibers inactivated both MS2 and PhiX174. (C) 2015 Elsevier B.V. All rights reserved.