Ticks are important vectors of pathogenic bacteria that cause diseases in both humans and animals. Analysis of tick microbiota via massive sequencing allows rapid and comprehensive identification of almost all bacteria inhabiting ticks. This has improved the detection of emerging pathogens and has helped define their relationship with public health. In Mexico, the brown dog tick (Rhipicephalus sanguineus sensu lato) is a public health problem, especially in northeast Durango. In the present study, the bacterial microbiota of this tick was determined using third-generation massive sequencing (PacBio, V1–V9 region of the 16S rRNA gene); bacteria with pathogenic potential that are transmitted by salivation and those that can be transmitted by accidental regurgitation of the parasite were also identified. In 2024, 60 dogs were searched for unfed ticks; then, 15 groups of female ticks and 15 groups of male ticks were formed, with each group consisting of 30 individuals. DNA was extracted from each tick pool, and the complete 16S rRNA gene was amplified (PacBio). Bioinformatics analysis was performed in QIIME2 (Quantitative Insights into Microbial Ecology) to obtain amplicon sequence variants (ASVs). Alpha and beta diversity metrics, as well as statistical analyses, were performed to test for differences between the microbiota of females and males. The bacterial taxa were classified into 21 phyla, 24 classes, 81 orders, 137 families, 339 genera, and 565 species. The male microbiota presented a significantly greater number of ASVs and a greater phylogenetic diversity index (FaithPD). Additionally, the unweighted UniFrac metric was significantly different between the sexes. The endosymbiont Coxiella mudrowiae was significantly more abundant in females, and Ehrlichia canis was more abundant in males. The pathogens E. canis and Anaplasma platys (transmitted by salivation) were detected, as well as 75 species of potential pathogens recorded in this tick that could enter the host in case of accidental regurgitation of the parasite (e.g., Staphylococcus, Streptococcus, Acinetobacter, Corynebacterium, Proteus, Clostridum, Klebsiella, Bacillus, Escherichia, Fusobacterium, and Pasteurella). It is necessary to continue analyzing the microbiota of ticks through massive sequencing for the benefit of public health and to establish new alternatives for controlling these parasites.