The prevalence of Anaplasma, Ehrlichia, Rickettsia and Babesia/Theileria species was analysed in questing and feeding adult ticks in Sicily. A total of 678 ticks were collected and analysed in this study. Of these, 29 were questing ticks and 649 were collected from infested cattle, sheep, goats or dogs. Tick species analysed included Rhipicephalus bursa, R. turanicus, R. sanguineus, Hyalomma lusitanicum, H. marginatum, Dermacentor marginatus, Ixodes ricinus, R. (Boophilus) annulatus and Haemaphysalis punctata. With the exception of R. annulatus and H. punctata for which only eight and 15 ticks were analysed, respectively, all tick species were found to be infected. Most ticks were found to be infected with a single pathogen genus. Data obtained from questing ticks was analysed to test for differences between tick species in the prevalence of infection for different pathogens. These preliminary results suggested that the most important vectors of pathogens that may affect human and/or animal health in Sicily are R. turanicus for Anaplasma spp. and D. marginatus for Rickettsia spp. For Ehrlichia spp. and Babesia/Theileria spp., R. turanicus/D. marginatus and H. lusitanicum may be the most important vectors but additional studies are needed to confirm these results.