Ultrasound (US) is a technology that enables microbial inactivation through cavitation-induced cell wall disruption, preserving food safety and quality. This study evaluated the impact of US parameters, including time, temperature, and surrounding media (saline solution and lipid parenteral emulsion) on Escherichia coli and Staphylococcus aureus reduction. Microbial survival was quantified via plate counting, and inactivation kinetics were modeled using GInaFiT. Microbial reductions ranged from 0.05 to 6.10 Log₁₀ CFU/mL, with E. coli showing greater susceptibility than S. aureus. The highest reduction (6.10 Log₁₀ CFU/mL) was observed for E. coli in the emulsion after 5 min at uncontrolled temperature, while S. aureus exhibited lower susceptibility (3.92 Log₁₀ CFU/mL). The Weibull model provided the best fit, highlighting the non-linear nature of microbial inactivation. The US presents a promising alternative for microbial control in food and pharmaceutical applications. Future research should optimize treatment conditions, understand microbial resistance mechanisms, and integrate the US with other hurdle technologies to enhance efficiency. In addition, studies about the US’s scalability for the pharmaceutical industry could widespread its implementation in that sector.