A study on tick fauna and tick-borne pathogens was undertaken in Pianosa, an island in the Tuscany Archipelago that constitutes an important stopping and nesting point for migratory birds. Ticks were removed from feral cats and a few terrestrial birds, and host-seeking ticks were collected by dragging. A total of 89 ticks were found on animals: 57 Ixodes ventalloi Gil Collado, 1936 and 32 Ixodes acuminatus Neumann, 1901. Host-seeking ticks were 354 Hyalomma spp. larvae and 18 Hyalomma spp. adults, identified as Hyalomma marginatum C.L. Koch, 1844 (n = 11) and 7 Hyalomma detritum Schulze, 1919 (n = 7). A sample of adult ticks was subjected to molecular analyses to look for Rickettsia spp. and Borrelia burgdorferi sensu lato (s.l.). Sequence analysis of the 5S-23S intergenic spacer region and OspA gene of B. burgdorferi s.l.-positive samples showed the presence of Borrelia spielmanii (n = 3; 3.7%, 95% confidence interval [CI] 0.08-10.4) and Borrelia valaisiana (n = 13; 13.6%, 95% CI 7.0-23.0) in Ixodes ticks from cats and terrestrial birds. Ixodes spp. were also infected by Rickettsia helvetica (n = 19; 23.4%, 95% CI 14.7-34.2). Finally, we detected Rickettsia aeschlimannii in 3 out of 12 host-seeking Hyalomma spp. adults tested (25%, 95% CI 5.5-57.2). Our study shows the presence of several tick-borne pathogens in Pianosa. Hyalomma spp. and Ixodes ticks other than I. ricinus seem to be involved in their epidemiological cycle, and birds could contribute to the pathogen dispersal along their migration routes. This is the first finding of B. spielmanii in Italy. We hypothesize the involvement of peridomestic rodents or hedgehogs in its maintenance in Pianosa.