The microflora in the periodontal pockets can affect the dental pulp and cause endodontic-periodontal lesions or retrograde pulpitis. Here we report an endodontic-periodontal lesion together with its bacterial profile. The lesion occurred in the maxillary right first molar of a 40-year-old woman who presented at our hospital complaining of a violent toothache since the previous night. Clinically, the tooth was caries-free and an electric pulp test showed it to be vital. The tooth showed signs of advanced periodontitis and the periodontal pocket was deep, reaching the apex of the palatal root. The clinical diagnosis was an endodontic-periodontal lesion with primary periodontal disease. Subsequent endodontic treatment comprised pulp extirpation and root canal filling, followed by periodontal treatment consisting of scaling and root planing. The tooth was finally restored with a full metal crown. No further signs of periodontal disease or periapical lesions have been observed to date. Bacteria were sampled from the root canal and periodontal pocket for a microbiological assessment using 16S rRNA gene-based PCR. Microbiologically, the profile of the bacterial species from the palatal root canal was similar to that from the periodontal pocket of the palatal root. Porphyromonas gingivalis, Fusobacterium nucleatum and Eikenella corrodens were detected in both samples. The occurrence of bacteria common to both sites in this patient further supports the proposition that periodontal disease is the definitive source of root canal infections. The present results suggest that a bacterial examination would be helpful in confirming and supporting the clinical diagnosis in such lesions.