Ticks transmit a greater variety of pathogenic microorganisms than any other arthropod group and are among the most important vectors of diseases affecting humans, livestock and companion animals. Ixodes holocyclus and Ixodes ricinus are the most important tick species from both a medical and a veterinary perspective in Australia and Eurasia respectively. However, little is known about microbial populations associated with indigenous Australian tick species. In the present study, high throughput 16S rRNA metabarcoding (amplicon sequencing) of the Australian paralysis tick I. holocyclus and the European Sheep tick I. ricinus was conducted. Initial analysis identified that ~99% of 16S sequences from I. holocyclus and I. ricinus ticks belonged to the tick intracellular endosymbiont Candidatus Midichloria mitochondrii. Relative copy-number of the Midichloria masked other bacterial communities and was impervious to switching 16S primers, sequencing platform or sequencing at greater depths. The development and use of a specific Candidatus M. mitochondrii blocking primer decreased the number of endosymbiontsequences by 95.8% in I. holocyclus and 96% in I. ricinus samples. This allowed identification of 199 and 94 bacterial genera in I. holocyclus and I. ricinus ticks, respectively. Of particular interest, a novel Candidatus Neoehrlichia sp. was identified in I. holocyclus. Recently, Candidatus Neoehrlichia mikurensis was identified as an emerging tick-borne pathogen of animal and human health importance. Our preliminary data, with a 94-95% sequence homology to Candidatus Neoehrlichia spp., indicates an Australian variant found within I. holocyclus. These results show that without blocking primers and the use of NGS molecular advances, many bacteria of potential medical and veterinary interest could go undetected.