The aqueous extract from the bark of Eucommia ulmoides serves as a rich source of bioactive compounds with numerous health benefits. The protocol here aims to explore the preparation of zinc oxide (ZnO) nanoparticles using the Eucommia ulmoides bark-mediated polyisoprene-rich aqueous extract. Meanwhile, the proposed protocol is associated with the preparation of wound healing material by easing the process. In addition, the wound-healing potential of the synthesized nanoparticles (Eu-ZnO-NPs) was evaluated using a simple scratch assay on a human umbilical vein endothelial cell (HUVEC) monolayer. After 24 h of treatment with Eu-ZnO-NPs, the cell proliferation and migration of HUVEC cells were assessed. At the end of the study, cell proliferation and migration were observed in scratched monolayer treated with different concentrations of Eu-ZnO-NPs, whereas poor cell migration and proliferation rates were observed in control cells. Of the chosen concentrations, 20 µg/mL Eu-ZnO nanomaterials showed better cell migration and enhanced wound healing potential.