Ornithodoros rostratus is an argasid tick distributed in Argentina, Bolivia, Brazil, and Paraguay. The objective of this study is to preliminarily characterize the composition and diversity of the associated bacterial microbiome, expanding knowledge about its microbial community. Ticks were collected in the central area of the Brazilian Pantanal (Nhumirim Ranch), state of Mato Grosso do Sul, central-western Brazil. Specimens were processed individually and subjected to surface disinfection (3× 1% sodium hypochlorite, 3× 70% ethanol, 3× sterile distilled water). DNA extractions were performed on three male specimens and six negative controls, in which all steps of the protocol were conducted. The obtained DNA was used to construct libraries targeting the V3–V4 hypervariable region of the 16S rRNA gene, which were sequenced on the Illumina NovaSeq 6000 platform (2 × 250 bp). Amplicon sequence variants (ASVs) were inferred using the DADA2 pipeline, and taxonomic assignment was performed using the SILVA database (v. 138.2). High-throughput sequencing generated a total of 962,554 reads, with 325,034 from male samples and 637,520 from extraction controls. The average sequencing depth was 106,950 reads per library, with a standard deviation (SD) of 4,708. After quality control, an average of 98,006 ± 5,368 sequences per library were retained. The phylum Actinomycetota was more abundant in the male specimens (87.33% ± 12.16), while Bacillota and Pseudomonadota showed average relative abundances of 5.24% ± 3.13 and 3.69% ± 4.82, respectively. The most representative families and genera were Brevibacteriaceae 35.26% ± 27.53 (Brevibacterium - 35.25% ± 27.52) and Pseudonocardiaceae 27.43% ± 22.19 [(Saccharopolyspora - 23.68% ± 23.26) and Arthrobacter (7.98% ± 1.94)]. Brevibacterium can play an important role in blood digestion in hematophagous arthropods. Saccharopolysporais an environmental bacterium known for its ability to produce bioactive natural products, including antibiotics. Arthrobacter comprises soil species associated with invertebrates as insects and ticks, demonstrating ecological functions, as it has the ability to use pyridone as its sole carbon source and resistance to desiccation and starvation. Our studies are still being completed. Therefore, the results presented here represent preliminary data.