Apical periodontitis, an inflammatory process around the apex of a tooth root, is primarily a sequel to microbial infection of the pulp space. The microbial flora is composed of a restricted group of the total oral flora, selected by environmental pressures of anaerobiosis, nutrition and competition with other species and inhabits the root canal as a biofilm of coaggregated communities in an extracellular matrix. The untreated infected canal is generally composed of a polymicrobial mix with approximately equal proportions of Gram-positive and Gram-negative species, dominated by obligate anaerobes. The type of microbial flora in the root-filled tooth with persistent apical periodontitis has very different characteristics. These infections are characterized by one or just a few species, predominantly Grampositive micro-organisms with an equal distribution of facultative and obligate anaerobes. Enterococcus faecalis has been a conspicuous finding in most studies. Because the primary aetiological problem is infection, endodontic treatment is directed at control and elimination of the root canal flora by working in a sterile way. Based on current knowledge, the best available method for obtaining clean, microbe-free root canals is by instrumentation with antimicrobial irrigation reinforced by an intracanal dressing with calcium hydroxide.Key words: Root canal flora, ecological niche, antibacterial treatment, root canal preparation, root canal irrigants.Abbreviations and acronyms: EDTA = ethylenediamine tetra-acetic acid; FTM = fluid thioglycolate medium; NaOCI = sodium hypochlorite solution; SEM = scanning electron microscope.