Most ultrasound inactivation studies conducted in food systems however use single frequency waves to generate cavitation. In a single frequency wave treatment, cavitation is limited and therefore compromises the biocidal efficacy of the process. Such limitation may be addressed by using multi-frequency ultrasound, and maximize cavitation yield (Feng et al., 2002 and Ohwada et al., 1995). This study was therefore conducted to characterize inactivation behaviors and establish inactivation parameters of some disease- and spoilage-causing microorganisms in an orange juice suspending medium. This study also traced the development of sublethal injuries in some of the test microorganisms as a result of multi-frequency ultrasound exposures. The influences of some microbial- and process-related variables on inactivation such as composite or cocktail inoculation, acid adaptation, and sensible heat propagation and equilibration within the suspending medium were also determined. The results of this study may be used in the establishment of multi-frequency ultrasound process schedules for orange juice and other similar products.