Electrospinning technique enabled us to prepare polymeric nanofibers. The current study was aimed to fabricate novel electrospun nanochitosan/poly(vinyl alcohol)/montmorillonite clay nanofibers by reactive electrospinning process and was carried out under electrical field strength of 22 kV with the flow rate of 0.55 mL/hr and the rotation speed of 1400 rpm. The particle size of the nanochitosan was measured using Dynamic Light Scattering technique and it was confirmed to be 100 nm. The synthesized nanofibers were characterized using FT-IR spectroscopy, X-ray diffraction, Differential scanning calorimeter, Scanning electron microscopy and Transmission electron microscopy. The antimicrobial property of the nanomat was also investigated using pathogens. The electrospun fibers’ diameter, being the key factor that determines the functional characteristics of an electrospun end product, was evaluated for the prepared mat which exhibits diameters below 100 nm from TEM technique. The blending of homopolymers was evidenced by FT-IR results. The diffraction peaks indicates the amorphous nature and the crystallite size was calculated using X-ray diffraction. The thermal stability of the mat was confirmed using Differential Scanning calorimeter. The morphology of nanofiber mat was investigated by Scanning electron microscopy. The preliminary studies supported that the mat prepared was a nanofiber mat exhibiting good antimicrobial property and it can be used for tissue engineering application in future.