This article demonstrates the use of the electrodeposition technique as a viable alternative for depositing uniform, ultra-smooth thin films of silk fibroin (SF). Herein, the electrodeposition of silk fibroin films from a homogeneous silk solution prepared by dissolving SF in formic acid (FA) solution is demonstrated. Ion electrodiffusion is used as the mechanism for the silk deposition. Optimizations were carried out to increase the β-sheet crystallinity, which in turn would enhance the inherent piezoelectric response of silk thin films, an exquisite property of silk that is seldom explored. The piezoelectric coefficient (d33) of electrodeposited silk thin film was experimentally figured out to be 8.39 pm/V. As a proof of concept, silk-based flexible nanogenerator was fabricated and tested successfully. Silk-based nanogenerator is able to produce a maximum open-circuit voltage of 1.02 V (peak–to-peak value) and short-circuit current of 0.8 mA at bending state. The device also exhibits good stability and reliability in terms of electrical outputs. © 2021 John Wiley & Sons Ltd.