Dental high-speed turbines and handpieces can take up and expel microorganisms during operation and thus need regular sterilization. This study established a method for validating devices used to sterilize high-speed turbines and handpieces. The air and water channels and turbine chambers were contaminated with suspensions of Streptococcus salivarius or endospores of Bacillus stearothermophilus. The effect of flushing and/or autoclaving performed by a new device combining both procedures was evaluated by counting the number of viable bacteria recovered from these devices. Further, the effect on clinically used handpieces was evaluated. In an initial experiment, the device partially reduced S. salivarius, and the endospores survived. In a second experiment, a 5 to 6 log reduction of S. salivarius in air and water channels was obtained. No growth was observed in clinically used high-speed handpieces, and both S. salivarius and endospores were eliminated from the turbine chambers. Thus, the method of validation proved capable of discriminating between different levels of bacterial reduction.