Nature can provide inspiration for the advancement of energy-harvesting technologies. This study describes a biomimetic nanofiber-based triboelectric nanogenerator (BN-TENG). Two-component electrospinning was used to prepare biomimetic nanofibers as the tribo-materials. The entire nanofiber mat (surficial nanostructures and internal biomimetic structures) were utilized to enhance the energy-harvesting performance of the BN-TENGs. Besides, charge retention on the mats was achieved via pressure treatment with a honeycomb mold. The optimized BN-TENG had the desirable power-generating performances: 1000 V of output voltage (with a load of 100 MΩ), and 364 μC m−2 of short-circuit transfer charge density. We also demonstrate the ability of the optimized BN-TENG to power electronics.