Biofilms are complex microbial communities that present serious contamination risks to our environment and health. In this study, atmospheric air plasma and airborne acoustic ultrasound technology were applied to inactivate Escherichia coli and Listeria innocua biofilms. Both technologies were efficient in controlling, or completely inactivating, the target bacterial biofilms. Viability and metabolic assays, along with microscopy analysis, revealed that atmospheric air plasma and airborne acoustic ultrasound damaged both the bacterial biofilm cells and its structural integrity. Scanning electron microscopy images highlighted the disruption of the biofilms and pore formation in bacterial cells exposed to both the plasma and acoustic treatments. Elevated reactive oxygen and nitrogen species in bacterial cells treated with atmospheric air plasma, demonstrated their primary role in the observed bacterial inactivation process. Our findings provide potential antimicrobial strategies to combat bacterial biofilms in the food and healthcare sectors.