Biotransformation is an eco-friendly and efficient method for enhancing the bioavailability of biopesticide. To increase the antifungal activity of the crude extract of Cynanchum wilfordii roots against barely powdery mildew, we performed biotransformation of wilfoside C1G using β-glucosidase (cellobiase from Aspergillus niger). The mixture (G sample) of partially purified wilfoside C1G and cynauricuoside A (K1G) was treated with β-glucosidase to remove a glucopyranosyl moiety. The enzyme completely converted C1G to C1N and K1G to K1N. Optimal conditions for enzymatic biotransformation of G sample were determined to be 10%ethanol, 1,555 μU β-glucosidase/ml, pH 5, and 45oC. In in vivo experiment, the G sample transformed by β-glucosidase showed stronger antifungal activity against barley powdery mildew than the non-treated G sample. These results suggest that β-glucosidase biotransformation can be applied to increase the antifungal activity of the crude extract of C. wilfordii roots against powdery mildews.