Well aligned crystalline zinc oxide (ZnO) nanowires (NWs) on ZnO/Au/Ti/Si substrates were grown by so-called “hydrothermal synthesis”. ZnO seed layers with different thicknesses ranging from 5 to 100 nm, achieved by controlling the deposition time, were prepared by radio-frequency sputtering, followed by a post-annealing treatment in air at 400 °C. The effects of deposition time and annealing treatment of ZnO seed layers on the subsequent growth of ZnO NWs were investigated using X-ray diffraction (XRD), atomic force microscopy (AFM), and scanning electron microscopy (SEM). The experimental results reveal that the quality and growth behaviors of ZnO NWs are strongly dependent on both the thickness and the heat treatment of the ZnO seed layers. This work is an optimization step of an easy, cost-effective, and industrially scalable process flow recently developed for the fabrication of a high performance, nanocomposite-based stretchable nanogenerator (SNG) on polydimethylsiloxane (PDMS) substrate. The morphological improvement of hydrothermally grown ZnO NWs may therefore lead to higher performance SNGs for the targeted application of mechanical energy harvesting, in order to supply flexible and wearable electronics.