This paper introduces a new approach of testing piezoelectric nanofibers as antibacterial mat. In this work, both Polyvinylidene fluoride (PVDF) and PVDF embedded with thermoplastic polyurethane nanofibers are synthesized as nanofibers mat via electrospinning technique. Then, such mat is analyzed as piezoelectric material to generate electric voltage under different mechanical excitations. Furthermore, morphological and chemical characteristics have been operated to prove the existence of beta sheets piezoelectricity of the synthesized nanofibers mats. Then, the synthesized nanofibers surfaces have been cyclically stretched and exposed to bacteria specimen. It has been noticed that the generated voltage and the corresponding localized electric field positively affect the growth of bacteria and reduces the formation of K. penomenue samples bacteria colonies. In addition, the effect of both stretching frequency and pulses numbers have been studied on the bacteria count, growth kinetics, and protein leakage. Our contribution here is to introduce an innovative way of the direct impact of the generated electric field from piezoelectric nanofibers on the reduction of bacteria growth, without depending on traditional anti-bacterial nanoparticles. This work can open a new trend of the usability of piezoelectric nanofibers through masks, filters, and wound curing mats within anti-bacterial biological applications.