Soybean isoflavones have been underestimated about their possible antifungal or antimycotoxigenic potential. In the present investigation, chemometric approaches were used such as the fractioned experimental design, the central composite rotatable design, and the Derringer’s desirability function to optimize a method to extract the isoflavones daidzein and genistein by ultrasound-assisted extraction. The optimized method was validated and applied to ten soybeans. The possible antimycotoxigenic abilities of isoflavones were evaluated by the inhibition of hydrolytic and oxidative enzymes. The multivariate procedures were efficient to determine the optimal extraction conditions, using daidzein and genistein concentration as responses. The best conditions were extraction time, 30 min; frequency, 55 kHz (36 kJ mL−1); hydrolysis time, 20 min; and hydrolysis temperature, 168 °C. The method was validated according to Eurachem guidelines and considered reproducible. The daidzein concentration was higher than genistein in soybeans. Low concentrations of aglycones were capable to inhibit fungal α-amylase (0.1 μg mL−1), highlighting genistein. Daidzein and genistein were able to inhibit oxidative processes in two strands, inhibition of oxidative enzyme and free radical propagation. These properties indicate that these isoflavones are able to contribute to avoid stress situations that could promote fungal contamination and manifestation of their mycotoxigenic potential.