Ultrasound (US)-based disinfection involves the disruption of cell membranes, oxidation of active free radicals, as well as hotspot heating, either individually or in combination. Various factors can affect US efficiency for inactivating microbes. However, only a few studies have discussed the use of US for microbial inactivation via response surface methodology. Here, we evaluated the potential of US for the disinfection of water supply or the elimination of drinking water residues. Moreover, the effects of US inactivation parameters, such as energy density, sonication time, and US device duty cycle on reduction in total bacterial (TB) count and total coliform (TC) count were investigated and optimized. The results indicated that the optimal inactivation condition was achieved at an energy density of 8.30 W/mL, a sonication time of 950 s, and a duty cycle of 0.7:0.3. Under optimal conditions, the experimental values of TB and TC inactivation efficiency were 47.26% ± 4.35% and 39.23% ± 2.27%, respectively, while the predicted values were 46.57% and 38.65%, respectively. The models developed here helped to predict the effectiveness of inactivation efficiency to a ‘sufficiently applicable’ extent. Under the optimized conditions, US has high potential as an effective disinfection method, as shown by energy efficiency analysis.