Enzymatic activity of potential interest in wine-making was studied for 182 non-Saccharomyces yeasts isolated from musts before and at the onset of fermentation at wine cellars operating under the La Mancha Appellation of Origin in Spain. Tests were carried out on plates containing differential substrates appropriate for each case (casein, gelatin, polygalacturonic acid, and arbutin) to determine whether each of the isolates exhibited proteolytic, polygalacturonase, and beta-glucosidase activities. Nearly 80% of the wild yeasts possessed one or more enzymes of biotechnological interest. Once the enzymatic activities of the isolates had been established, 69 of the isolates that exhibited pronounced enzymatic activity and 11 randomly selected isolates that were devoid of any activity were typed using PCR/RFLP, which gave 13 different molecular profiles. The isolates for each of the profiles were then identified by classical methods. The enzyme beta-glucosidase was linked to the species Metschnikowia pulcherrima, and polygalacturonase activity was common in most of the species identified. Proteolytic activity was observed in Pichia membranifaciens and in Metschnikowia pulcherrima. Typing revealed the possibility of intraspecific differences in Pichia membranifaciens, because six different molecular profiles with one or more shared restriction bands were recorded for that species.