The aim of this study was to investigate the inactivation of nonpathogenic Escherichia coli in nutrient broth and milk through the use of either ultrasound (US) alone or US combined with nisin (US + nisin) treatments. The E. coli cells were treated at 0 to 55°C, 242.04 to 968.16 W/cm2 for 0 to 15 min. The results showed that the inactivation of E. coli by US and US + nisin increased when the temperature, US power density, and treatment time were increased. The inactivation kinetics of E. coli in nutrient broth by US and US + nisin both conformed to linear models. The largest reductions of 2.89 and 2.93 log cycles by US and US + nisin, respectively, were achieved at 968.16 W/cm2 and at 25°C for 15 min. The suspension media of the E. coli cells influenced the inactivation effect of US, while the growth phases of E. coli cells did not affect their resistance to US. Under all experiment conditions of this study, the differences between US and US + nisin in their respective inactivation effects on E. coli were not obvious. The results suggested that nisin had either no effect at all or a weak synergistic effect with US and that the E. coli cells were inactivated mainly by US, thus indicating that the inactivation of E. coli by US is an “all or nothing” event.