The horn fly, Haematobia irritans, is one of the economically important pests of cattle. Use of insecticide is a major element of horn fly management programs. Growing concerns of insecticide resistance, insecticide residues on farm products and non-availability of new generation insecticides in the near future are serious issues of the organized livestock farming industry. Alternative horn fly control methods would reduce the use of insecticides and reduce the amount of insecticide residues on livestock products and boost the organic livestock-farming segment. The horn fly, an obligatory blood feeder, requires microbiota to supply additional nutrients and metabolize the blood meal. A culture-independent method, bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) has been used to classify the diverse bacterial microbiota in adult female horn flies, adult male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in these samples, which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with dipteran-pests affecting livestock using a culture-independent method. Several rumen, symbiotic and pathogenic bacteria associated with the horn fly were quantified. This is the first report of presence of Wolbachia in the horn fly of USA origin and is the first report of presence of Rikenella in an obligatory blood-feeding insect.