The effectiveness of a newly developed anti-cross contamination device for a dental air-turbine handpiece was tested. The handpiece with or without the anti-cross contamination device was contaminated with two bacterial strains, Staphylococcus aureus and Streptococcus mutans, as well as two bacteriophage strains, T2 and MS2. After contamination with these microorganisms, the handpieces were disinfected with glutaraldehyde or replaced with newly autoclaved ones. Residual microorganisms inside the handpiece or an air/water supply hose line were collected and counted after overnight cultivation. The anti-cross contamination device effectively reduced the contamination level of an air-turbine handpiece to that of the negative control. No microbial contamination in the air/water supply hose line was detected with this device.