Inoculation trials performed with three strains of yeasts, isolated from extra virgin olive oil, Williopsis californica 1639, Saccharomyces cerevisiae 1525 and Candida boidinii 1638, demonstrated that some yeast can lower the quality of the oil during storage. Laboratory tests highlighted a substantial increase in the total diglycerides and free fatty acids in the samples of oil inoculated with the lipase-producing strains of yeasts, W. californica 1639 and S. cerevisiae 1525, while in the samples of oil inoculated with the lipase-negative strain C. boidinii 1638 no differences were found in respect to the uninoculated control. The acidity of the extra virgin olive oil, inoculated with the lipase-producing strains W. californica 1639 and S. cerevisiae 1525, during 2 weeks of incubation at 30°C increased respectively from 0.62% to 1.50 and 1.62%, exceeding the limit of 0.8% established by current regulations for this commercial category of olive oil, while in the oil inoculated with the lipase-negative strain and in the uninoculated control, the acidity remained constant throughout. Furthermore, the two strains of lipase-producing yeasts also increased the concentration of the 1.3-diglyceride isomer in the oil lowering the values of the total 1.2-diglycerides/total 1.3-diglycerides ratio considered to be an important index of quality for an extra virgin olive oil. The lipolytic activity of lipase-producing strains W. californica 1639 and S. cerevisiae 1525 showed an optimum pH of 6 and 7.5 and an optimum temperature of 20°C and 30°C respectively. Nevertheless, the lipolytic activity was negatively influenced by glucose and polyphenols when the concentration was higher than 0.25% and 0.4% (wt/vol) respectively.