Tick-borne bacteria pose a significant threat to human and veterinary public health. Greece is a Mediterranean country with rich tick fauna and the most commonly detected tick-borne bacterial pathogens are members of the and species. The variable V2-V4 and V6-V9 regions of 16S rRNA gene of seven ticks belonging to four genera representative in Greece () were analysed using multiple primer pairs by next generation sequencing (NGS). Nine bacterial phyla corresponding to 95 families, 116 genera and 172 species were identified. Proteobacteria was the predominant phylum in five of the seven ticks, followed by Actinobacteria, which predominated in two ticks. The tick-borne bacteria included and species, while " Midichloria mitochondrii" were detected in high abundance in ticks and less in ; -like endosymbionts were detected in , , and less in ticks. Co-infections with and were also observed. 16S rRNA NGS is a powerful tool to investigate the tick bacteriome and can improve the strategies for prevention and control of tick-borne diseases.