The present study aimed to identify ticks collected from road-killed wildlife species retrieved in several localities of southern Italy and to assess the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. DNA in ticks. Collections were carried out from January 2000 to December 2009 on wild animals found dead within the territories of 11 municipalities from three regions (i.e., Apulia, Basilicata, and Calabria). In total, 189 carcasses of wild animals belonging to 10 species were checked for tick infestation, and 40 animals belonging to seven species were found parasitized. One hundred and twenty-five ixodid ticks (11 larvae, 14 nymphs, 77 males, and 23 females) were collected and identified as belonging to nine species, namely Dermacentor marginatus, Haemaphysalis erinacei, Hyalomma marginatum, Ixodes acuminatus, Ixodes canisuga, Ixodes hexagonus, Ixodes ricinus, Rhipicephalus bursa, and Rhipicephalus turanicus. None of the 36 tick specimens tested by PCR was positive for tick-borne pathogens. The results add new information on the tick fauna associated with wild animals in Italy, reporting new tick-host associations. Further field studies are still needed to ascertain the suitability of certain wildlife species as hosts for some tick species, particularly for those implicated in the transmission of pathogens to domestic animals and humans. Finally, from a conservation perspective, it would be interesting to assess whether these wild animals (e.g., Lepus corsicanus) are exposed to tick-borne pathogens, investigating the possible implications for their health and behavior.