The aim of this study was to identify bacteria from the infected root canals of teeth with chronic apical periodontitis, and to evaluate the antibacterial effect of 0.2% chlorhexidine (CHX), as an irrigant, in reducing the microbial flora 48h after root canal preparation. A total of 44 subjects were randomly divided in the experimental group and the control group. The first bacterial samples from all root canals were obtained in the beginning, before any treatment. During mechanical instrumentation, root canals were irrigated three times, with 0.2% CHX in the experimental group, and with saline solution in the control group. All canals were dried and temporarily sealed with zinc oxide-sulfate cement. After 48 h the second samples were obtained. Bacterial samples were subjected to microbiologic processing. The study indicates that 0.2% CHX is significantly effective in reducing the microbial flora, and could be used as an irrigant solution.