The aim of this study was to investigate the influence of thermal treatment and ultrasound on the activity of β-glucosidase and lipoxygenase, enzymes that determine the phenolic composition and sensory profile of virgin olive oil. Enzyme activity was determined spectrophotometrically in model systems consisting of commercial enzymes and their substrates. Thermal treatment was performed by tempering the enzymes and substrates at temperatures between 15 and 40 °C. Enzyme activity was measured 1 min after reaction and again after the additional incubation of the reaction mixture at 25 °C for 30 min to simulate the behavior of the enzymes during the malaxation process. Ultrasonic treatment of the model solutions was performed at 128, 320, and 640 W of the ultrasonic bath power for 1, 5, and 12 min. Enzyme activity was determined immediately after treatment and again after incubation at 25 °C for 30 min. The higher temperatures during thermal treatment increased the activity of both enzymes. During ultrasound treatment, the activity of both enzymes was positively affected by its duration. The higher power of ultrasound was a better choice for β-glucosidase and the lower one for lipoxygenase. The stimulation of enzyme activity by the studied techniques resulted in an acceleration of enzymatic reactions during the additional incubation, suggesting that the malaxation process could be shortened in virgin olive oil production.