Rodents are key reservoirs of zoonotic pathogens and play an important role in disease transmission to humans. Importantly, anthropogenic land-use change has been found to increase the abundance of synanthropic rodents, particularly rodent reservoirs of zoonotic disease. Anthropogenic environments also affect the microbiome of synanthropic wildlife, influencing wildlife health and potentially introducing novel pathogens. Our objective was to characterize the microbiome and investigate the prevalence of zoonotic bacterial pathogens in synanthropic rodents in native and anthropogenic environments to better understand their role in pathogen maintenance and transmission. We sampled wild Peromyscus mice in agricultural and undeveloped landscapes and forest and synanthropic habitat in Minnesota, USA and conducted 16S amplicon sequencing using long-read Nanopore sequencing technology on fecal samples to characterize the rodent microbiome. We compared community composition and diversity between habitats and screened for the presence of putative pathogenic bacteria species. Microbiome community composition differed significantly between agricultural and undeveloped landscapes and forest and synanthropic habitat while microbiome richness, diversity, and evenness were lower in undeveloped-forest habitat compared to all other habitats. We detected overall low abundance and diversity of putative pathogenic bacteria, though the greatest number of pathogenic bacteria were detected in the agricultural-forest habitat. Our findings show that rodent microbiome community composition differs across landscapes and habitat types but suggest that landscape-level anthropogenic factors may be most important to predict zoonotic pathogen abundance. Ultimately, understanding how anthropogenic land-use change and synanthropy affect rodent microbiomes and pathogen prevalence is important to managing transmission of rodent-borne zoonotic diseases to humans.