Abstract: PURPOSE To evaluate the influence of different root canal instrumentation size on disinfection of intracanal microbe of dental root canal. METHODS 368 extracted human anterior teeth with single straight root were randomly divided into 8 groups of 46 roots in each. They were instrumented with K3 Ni-Ti files as follows: group A1 and group B1(#25/0.06), group A2 and group B2(#30/0.06), group A3 and group B3(#35/0.06), group A4 and group B4(#40/0.06). After being prepared and sterilized by autoclaving, group A was inoculated with Enterococcus faecalis, and group B was inoculated with Candida albicans. All groups were irrigated with Er:YAG laser combination of 3% NaOCl, 17% EDTA and 0.9% saline, and then the numbers of microbe on the surface of root canal walls were counted after the treatment, the absolute reduction of counting colony forming units(CFUs) and the relative residual rate of CFUs in the individual group was determined. The Date was analyzed with GraphPad Prism 5.02 software package by one-way analysis of variance. RESULTS Levels of disinfection on E.faecalis and C.albicans increased when root canals were enlarged; #40/0.06 showed the best disinfection, #35/0.06 showed a significantly better disinfection than #30/0.06 and #25/0.06. Substantial reduction of microbe was obtained in #35/0.06 and #40/0.06 compared with #25/0.06 and #30/0.06(P＜0.05).CONCLUSIONS Within the root canal size of #25/0.06-#40/0.06, under the conditions of Er:YAG laser combination of 3% NaOCl, 17% EDTA and 0.9% saline, it was concluded that the reduction of E.faecalis and C.albicans of the anterior straight root canals could be predicted by increasing the root canal instrumentation size large than #30/0.06.