In this study, we investigated the effects of seed layer thickness on the structural and piezoelectric properties of ZnO nanowires (ZNWs) and evaluated their energy harvesting performance. ZNWs with seed layer thicknesses of 100, 200, 300, and 400 nm were synthesized and characterized. The results show that a ZNW with a 300 nm seed layer (ZNW-300) has the highest aspect ratio, resulting in excellent piezoelectric performance with a piezoelectric charge coefficient (d33) of 10.5 pm/V and a peak-to-peak voltage output of 4.6 V. The energy harvesting performance was further verified through a 150 μF capacitor charging test, which successfully charged to 3.5 V in 12 s, generating a power output of 76.56 μW. These findings highlight the potential of ZNW-based piezoelectric nanogenerators for self-powered applications, including wearable electronics, wireless sensor networks, and sustainable energy harvesting systems.