Ticks are important vectors of zoonotic pathogens, and the presence of these pathogens can be influenced by factors such as the tick microbiome, which, conversely, is shaped by environmental conditions and the presence of tick-borne pathogens, as demonstrated in several studies conducted under controlled conditions. However, the extent of these influences under natural ecological conditions remains underexplored. In this study, we investigated the diversity of the microbiome and the prevalence of pathogens in Ixodes ricinus nymphs across three distinct Belgian ecoregions: Sandy Loam, Condroz, and Ardennes. Using real-time quantitative PCR (qPCR) and Oxford Nanopore 16S rRNA sequencing, we assessed how geography and pathogen presence influence tick-associated microbial communities. Our results revealed significant regional differences in microbiome composition and pathogen prevalence. Borrelia burgdorferi sensu lato (s.l.) was most prevalent in the Ardennes, while Anaplasma phagocytophilum was more common in the Sandy Loam region. Endosymbionts such as Midichloria mitochondrii and Spiroplasma ixodetis also exhibited distinct geographic distributions. Network analysis identified potential pathogen-microbiota interactions, with certain bacterial taxa showing positive or negative associations with specific pathogens. Moreover, microbiome composition was influenced not only by ecoregion but also by pathogens such as Rickettsia helvetica, suggesting that its colonization may actively shape microbial community structure, potentially through competition or facilitation mechanisms. Additionally, microbiome network robustness varied across ecoregions, highlighting the role of ecological context in shaping microbial interactions within ticks. These findings underscore the complex interplay between geography, pathogen presence, and microbial diversity in ticks, emphasizing the need to consider ecological variability when studying tick-borne disease dynamics. Understanding these interactions could inform microbiome-based strategies for vector control and disease prevention.