This repository contains supplementary material derived from the article “Bacterial composition of Polygenis (Polygenis) bohlsi bohlsi (Wagner, 1901) (Siphonaptera: Rhopalopsyllidae) associated with Thrichomys fosteri (Rodentia: Echimyidae) in the Brazilian Pantanal Wetland”, and corresponds to the microbiome analysis based on the V3–V4 region of the 16S rRNA gene from flea specimens collected from the rodent Thrichomys fosteri in the Brazilian Pantanal wetland. The supplementary material includes: (i) detailed descriptions of the molecular assays used for the detection and characterization of Bartonella spp. and Wolbachia spp., including target genes, primer and probe sequences, amplification conditions, amplicon sizes, and references (Tables SM1 and SM2); (ii) molecular identification results and BLASTn analyses of amplicons obtained from flea specimens positive for Bartonella spp. (Table SM3); (iii) identification of analyzed samples and corresponding GenBank accession numbers for the generated sequences (Table SM4); (iv) molecular results and BLASTn analyses of samples positive for Wolbachia spp. (Table SM5); and (v) a rarefaction curve based on bacterial ASVs demonstrating that sequencing depth was sufficient to capture the majority of bacterial diversity across samples (Figure SM1).