We used Wilderlab eDNA kits to collect eDNA samples in a creek colloquially known as Cañada del Puerto Creek (Hydrologic Unit Code 12 number 180600140201) on Santa Cruz Island, California, in May 2025. Samples were sent to Wilderlab for metabarcoding analysis using their comprehensive panel of assays to amplify DNA from a variety of taxa, including vertebrates (fish, mammals, reptiles, amphibians, and birds), crustaceans, and freshwater insects. This dataset is the metabarcoding results, which includes exact sequent variants (ESVs) and their taxonomic assignments found in each sample using each assay.