Our objective is to analyze the constitution and drug resistance of pathogenic microorganisms in infected root canals. 69 patients with chronic periapical periodontitis were selected, the tooth root canals of which were collected. There were 15 cases (group A) with root canal infection after root canal treatment failure, 18 cases (group B) with root canal infection after dental crown restoration, 16 cases (group C) with root canal infection after pulpectomy and 20 cases (group D) with root canal infection after no treatment. The detection rates of bacteria in group A, B, C and D were 93.33%, 94.44%, 87.50% and 90.0% respectively, the detection rates were significantly higher than the detection rates of archaebacteria, which were statistically different (P<0.05); the detection rates of three actinomycetes including A. viscosus, A. naeslundii and A. israelii were 80%, 20% and 26.67% in group A which were all higher than group B, C and D; the detection rate of Enterococcus faecalis in group A was 73.33% which was obviously higher than group B, C and D, there were significant differences between groups (P<0.05). There were 8 cases with symptoms in mixed infection group with percentage of 80%, 26 cases with symptoms in the simple bacterial infection group with percentage of 49.06%, 2 cases with symptoms in the simple archaebacterial infection with percentage of 33.33%, the occurrence rate of symptoms in the mixed infection group was obviously higher than the other groups and there were significant differences between groups (P<0.05). After the root canal is infected by microorganism, there may be with symptoms or without symptoms. The infection microorganisms are mainly actinomycetes and Enterococcus faecalis, there are different infection microorganisms in different types of root canal infections.