The bacterial phylum Synergistetes consists of Gram-negative anaerobes. Oral Synergistetes are divided in two main clusters, namely A and B. Increasing evidence demonstrates their involvement in etiology of oral infections, including apical periodontitis. This condition causes bone loss around the apex of the tooth, subsequent to pulp inflammation (pulpitis). Although the presence of Synergistetes has been confirmed in endodontic infections by molecular methods, these have not been morphologically identified in the affected apical region, and their prevalence among different endodontic infections has not been determined. Therefore, the aim of this study was to evaluate the prevalence, levels and morphology of oral Synergistetes clusters A and B, in apical root canal samples obtained of teeth with irreversible pulpitis, pulp necrosis and apical periodontitis, or previously root-filled teeth with apical periodontitis. For their detection, fluorescence in situ hybridization and epifluorescence microscopy were used. Synergistetes cluster A was not detected in pulpitis, but was found in both apical periodontitis groups, more frequently and at higher ranges in teeth which were previously root filled. Microscopically, they appeared as straight or slightly curved long rods. Synergistetes cluster B was not detected in any of the cases. Fusobacteria and Actinomyces, which are well-established taxa in endodontic infections, were detected more frequently and at higher ranges than Synergistetes. In conclusion, Synergistetes cluster A constitutes part of the mixed apical microbiota in apical periodontitis, and may be involved in its pathogenesis.