Antimicrobial polyvinylidene fluoride (PVDF) membrane modified by dodecyltrimethyl ammonium bromide (DTAB) has been electrospun using simple one-step technology, where the modifying agent DTAB is dissolved in spinning solution. X-ray photoelectron spectroscopy and electrokinetic analysis confirm reliably the presence of DTAB on the nanofibers surfaces; electrokinetic analysis shows the changes of zeta potential due to modification by DTAB. X-ray diffraction shows that electrospinning converts the part of phase (approximate to 40%) present in PVDF powder into phase with all trans (TTT) zigzag chains conformation in PVDF electrospun membrane. Surface modification does not affect the phase composition of PVDF nanofibers, just only leads to lower crystallinity (smaller size of crystallites) in PVDF nanofibers. DTAB causes the curling of fibers and their aggregation, what completely changed the membrane structure. DTAB-modified membrane exhibits antibacterial properties against Staphylococcus aureus subsp. Aureus. Concentration of 0.5 wt% DTAB in spinning solution causes partial inhibition of bacterial growth only, while 1.0 wt% concentration leads to complete inhibition.