Ultrasound technology has been studied by food researchers as an alternative method for thermal processing. The use of ultrasound as a way to inactivate and/or activate enzymes has been widely studied at low frequencies (20–40 kHz), however, little research on the effect of high frequencies has been reported. Thus, the effect of high and low frequency ultrasound on commercial horseradish peroxidase with a concentration of 0.005 mg mL−1 is described. Experiments were performed for 60 min using 20, 378, 583, 862, 995, 1144 and 1175 kHz ultrasound at power levels (acoustic energy) between 2.1 and 64 W. Residual activity was monitored using a spectrophotometric method and data analysis was performed using ANOVA. A significant enhancement of enzyme inactivation (p < 0.05) was observed at each frequency with an increase of sonication time and power. Inactivation of peroxidase by ultrasound followed first order kinetics and an increase of the rate constant with the power applied was observed for all the frequencies studied. Overall, low frequency (20 kHz) and low power are not effective on the enzyme inactivation and the level of residual activity remained high. The use of 378 and 583 kHz (48 W) is particularly effective for complete enzyme inactivation.