A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces. However, the impact of the aerosols on the viability of bacteria during treatment has never been evaluated. In this study, the effects of high-speed CO2 aerosols, a mixture of solid and gaseous CO2, on bacteria viability was studied. It was found that when CO2 aerosols were used to disperse biofilms of Escherichia coli, they led to a significant loss of viability, with approximately 50% of the dispersed bacteria killed in the process. By comparison, 75.6% of the biofilm-associated bacteria were viable when gently dispersed using Proteinase K and DNase I. Indirect proof that the aerosols are damaging the bacteria was found using a recombinant E. coli expressing the cyan fluorescent protein, as nearly half of the fluorescence was found in the supernatant after CO2 aerosol treatment, while the rest was associated with the bacterial pellet. In comparison, the supernatant fluorescence was only 9% when the enzymes were used to disperse the biofilm. As such, these CO2 aerosols not only remove biofilm-associated bacteria effectively but also significantly impact their viability by disrupting membrane integrity.