A patient with an unusual endo-periodontal lesion, without coronal decay or damage, likely caused by a deep periodontal lesion with subsequent endodontic bacterial migration, required medical care. Next-generation sequencing (NGS) was used to assess the endodontic microbiota in vestibular and palatal canals after tooth extraction, evidencing a predominant population (Fusobacterium nucleatum) in one endodontic canal, and a mixed bacterial population with six major populations almost equally distributed in the other endodontic canal (F. nucleatum, Porphyromonas gingivalis, P. endodontis, Parvimonas, Peptostreptococcus stomatis, Prevotella multiformis). These data could suggest different, separated ecologic niches in the same endodontic system, with potentially different pathogenicity levels, clinical manifestations and prognoses for every single canal of the same tooth.