Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p > 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.