A total of 17 Pichia (Wickerhamomyces) isolates obtained from enological ecosystems in the Utiel-Requena Spanish region were characterized by physiological (using API 20C AUX strips and ID Yeast Plus System miniaturized identification systems) and molecular (PCR-RFLP and sequencing) techniques as belonging to the species P. fermentans, P. membranifaciens, and W. anomalus. Data support the reclassification of P. anomala as Wickerhamomyces anomalus. In order to characterize their enzymatic abilities, xylanase, β-glucosidase, lipase, esterase, protease, and pectinase qualitative and quantitative assays were made. Wickerhamomyces anomalus and P. membranifaciens were the most interesting species as a source of enzymes for the winemaking industry. Glycosidase enzymes had a high degree of tolerance to high levels of glucose and ethanol, making them of great interest for enological use.