Endodontic bacterial infection is the most often cause of apical periodontitis (AP). In some patients, inflammatory odontogenic cyst of the jaw, so called radicular cyst (RC), may develop. Analysis of microbiota in tooth root canal is limited by its morphology, the transition of microbiota from apex into RC is possible. The aim of this pilot study was to compare bacteriome of dental plaques, teeth affected by AP and cystic fluids from RC. Swabs of dental plaque from tooth AP and cystic fluid from 6 adult patients with radiographically and histopathologically verified finding of RC were obtained. From the reason of RC extirpation, the affected tooth was extracted in each patient and then crushed in in sterile capsule in cryomill. Microbial DNA was isolated from all three matrices by QIAamp DNA Mini Kit (QIAGEN) and used for PCR amplification of hypervariable 16S rRNA segments. Libraries for subsequent Next-generation sequencing on Illumina NovaSeq instrument was prepared according to the standard Illumina 16S metagenomic protocol. Alpha diversity based on amplicon sequence variant was statistically decreased in cystic fluid than in dental plaque and tooth with AP (p < 0.05 and p < 0.01, respectively). In comparison of individual matrices, there were significant differences in bacterial abundances (p ? 0.001), specifically in Rothia, Streptococcus, Capnocytophaga, Leptotrichia, and Veillonella sp. Cystic fluids had higher percentage in relative abundance of Treponema, Tannarella, Prevotella, and Porphyromonas sp. than teeth affected by AP and their dental plaques, respectively. In conclusion, RC might be polymicrobial originated. Non-sterile cystic fluid from RC contains these DNA from anaerobic periodontal pathogens, which are found also in the tooth affected by AP in the same patient. Acknowledgemets: The study was supported by the Ministry of Health of the Czech Republic, grants No. NU20-08-00205, and by a project funded by the University Hospital Brno, Ministry of Health, Czech Republic – RVO (FNBr, 65269705). This publication has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 857560. This publication reflects only the author´s view and the European Commission is not responsible for any use that may be made of the information it contains. The authors also thank the Research Infrastructure RECETOX RI (No LM2018121) and the project CETOCOEN EXCELLENCE (No CZ.02.1.01/0.0/0.0/17_043/0009632) financed by the Ministry of Education, Youth and Sports for supportive background.