The inactivation of Escherichia coli is one of the major issues in the food industry. The present study focuses on the application of a combined microwave-ultrasound system for the optimization of the inactivation of Escherichia coli ATCC 25922 in an orange juice drink. Using response surface methodology (RSM), trials were planned with a Box–Behnken Design (BBD) to maximize the impact of microwave power (A: 300–900 W), microwave treatment time (B: 15–35 s), and time of ultrasound (C: 10–30 min) on E. coli inactivation. Analysis of variance (ANOVA) was carried out and E. coli inactivation was expressed with a mathematical equation depending on the factors. The results showed that both the microwave treatment time and the time of ultrasound were effective as independent variables in eliminating the E. coli strain. However, the effect of these two variables, ultrasound and microwave exposure time, in combination was significantly greater than when examined separately. RSM modeling determined that optimal treatment conditions include 900 W microwave power, 33 s microwave treatment time, and 20 min time of ultrasound to achieve an 8-log reduction of E. coli, constituting total inactivation. The results of this study showed that ultrasound-microwave treatment is a potential alternative processing method for an orange juice beverage.