Today, people tend to consume products with high nutritional value. Ultrasound-assisted pasteurization is one of alternative methods for thermal pasteurization. In this study, red grape juice samples were treated by ultrasonication at different powers (10, 105, and 200 W), temperatures (0, 30, and 60 °C), and times (2, 6, and 10 min). After optimization by response surface methodology (Box-Behnken method), antioxidant potency, pectin methyl esterase activity, and microbial contamination (total count of microorganisms, coliforms, molds, and yeasts) of the red grape juice treated by the optimized ultrasonication were compared to those achieved by thermal pasteurization (at 90 °C for 30 sec) as control. By increasing power from 10 to 200 W, temperature from 0 to 60 °C, and time from 2 to 10 min, antioxidant potency, activity of pectin methyl esterase, and microbial population decreased in the samples, significantly. The optimum condition of ultrasonication to achieve the least microbial growth (non-detectable), and the highest antioxidant potency (IC₅₀ of 62.3062 µl/ml) and pectin methyl esterase activity (0.0568 PEU/ml) was predicted at 180 W, 42 °C, and 8 min. After optimization, there was no significant difference between microbial contamination in the sample treated by ultra­sonication and the thermally pasteurized red grape juice, but higher antioxidant potency and pectin methyl esterase activity was observed in the ultrasound-treated sample than control. Therefore, grape juice can be pasteurized by ultrasonication instead of thermal process, through which bioactive compounds are better preserved without safety concern in term of microbial contamination.