Plant-derived nanovesicles (PDNVs) were isolated from Ginkgo biloba seed homogenate using differential ultracentrifugation (DUC) followed by density gradient ultracentrifugation (DGUC) with linear and non-linear iodixanol gradients. Nanoparticle tracking analysis (NTA) and cryo-transmission electron microscopy (cryo-TEM) characterized vesicle size, concentration, and morphology. Untargeted mass spectrometry profiled the protein content of distinct PDNV fractions. Ginkgo PDNVs formed a heterogeneous population, including single- and double-bilayer vesicles >50 nm. Proteomics revealed seed storage proteins (legumin, ginnacin) and membrane-associated ATPases, HSP90, catalase, PEPC, and eEF1A. Ginkgo seed-derived PDNVs exhibit promising vascular protective and anti-inflammatory properties, supporting their potential as safe, multifunctional agents for endothelial modulation.