Water is one of the most essential resources for the survival of human beings and all other living things. For the point of daily use, water sterilization has enormous social and economic significance, especially for remote and undeveloped areas. Here, we developed a self-powered water sterilization device, which consists of a rotating-disk freestanding triboelectric-layer mode triboelectric nanogenerator (RF-TENG), a voltage-multiplying circuit, and a water droplet control system. The output voltage of the RF-TENG is boosted by a voltage-multiplying circuit and then utilized to charge water droplet. When the rotation rate of the RF-TENG is 300 rpm, the output voltage of a six-fold voltage-multiplying circuit can reach 9319 V, and a 62.50 μl water droplet can be positively charged to 6320 nC at the flow rate of 0.31 ml min−1. The charge density and electric filed of the water droplet can reach 101.12 nC μl−1 and 11.28 kV cm−1, respectively. The charged water droplet can kill E. coli and S. aureus quickly and efficiently through electroporation mechanism. With the advantages of low cost, simple in fabrication and usage, portability, and etc, the self-powered water sterilization device has wide application prospects in remote and undeveloped areas.