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Adult house flies (Musca domestica L.) are important reservoirs and mechanical vectors of bacteria in livestock operations. House fly bacterial communities are influenced by their local environment, yet a comprehensive understanding of bacterial diversity, pathogen prevalence, and bacterial source is not fully understood. We characterized bacterial communities from adult female house flies and associated manure samples from beef and dairy cattle farms in Kansas, Oklahoma, and Texas over four months (July-October). Bacterial community composition in flies and manure reflected the local environment, and house flies shared the majority (≥ 99%) of bacterial taxa with manure. The variability of bacterial diversity was greater among individual fly (species richness range: 48-1747) samples than manure (species richness range: 345-1162). Temporal variability of fly bacterial diversity was observed within each farm type. Bacterial taxa of veterinary and medical importance such as Corynebacterium, Turicibacter, Staphylococcus, Streptococcus, and Acinetobacter were highly prevalent in flies, constituting core bacterial communities. The prevalence of bacterial taxa associated with bovine keratoconjunctivitis (IBK) and bovine respiratory disease (BRD) was higher in flies than in manure and prevalence varied monthly. This study underscores the crucial role house flies play as carriers of cattle pathogens, contributing to their dissemination among animals and to off-site locations, where they pose a threat to surrounding communities and agricultural operations., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)