The principal aim of this study was to describe the microbiota of the hematophagous ectoparasites collected from migratory birds in Italy, mainly in the north west of the country (Piedmont region), by means of high-throughput 16S rRNA barcoding. Based on the rationale of describing the active microbiota, we used the total RNA in order to characterize only the actively living bacteria, without bias given by the DNA carried over from dead prokaryotic cells. Samples whose reads were classified as potentially pathogen bacteria were further processed for confirmation and characterization by molecular tests.