BackgroundHippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria.MethodsLouse flies were collected from naturally infested cattle (n= 25), sheep (n= 3), and red deer (n= 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened forBorreliaspp.,Bartonellaspp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida andBartonellaspp. were further characterized by phylogenetic and haplotype networking analyses.ResultsA total of 282 hippoboscid flies corresponding to three species were identified:Hippobosca equina(n= 62) collected from cattle,Melophagus ovinus(n= 100) from sheep andLipoptena cervi(n= 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual.BartonellaDNA was detected in 36.9% of the louse flies.Lipoptena cerviwere infected with 10 distinct and previously unreportedBartonellasp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description ofTrypanosomasp. inH. equina. Anaplasmataceae DNA (Wolbachiaspp.) was detected only inM. ovinus(16%), while < 1% of the louse flies were positive forBorreliaspp. and Filarioidea. All hippoboscids were negative for Piroplasmida.ConclusionsMolecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g.Bartonellaspp.) and the first report ofTrypanosomasp. inH. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05810-4.