Corsica is a mountainous French island in the north-west of the Mediterranean Sea presenting a large diversity of natural environments where many interactions between humans, domestic animals and wild fauna occur. Despite this favourable context, tick-borne pathogens (TBPs) have not systematically been investigated. In this study, a large number of TBPs were screened in ticks collected over a period of one year from domestic and wild hosts in Corsica. More than 1,500 ticks belonging to nine species and five genera (Rhipicephalus, Hyalomma, Dermacentor, Ixodes and Haemaphysalis) were analysed individually or pooled (by species, gender, host and locality). A real-time microfluidic PCR was used for high-throughput screening of TBP DNA. This advanced methodology enabled the simultaneous detection of 29 bacterial and 12 parasitic species (including Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia and Theileria). The Crimean–Congo haemorrhagic fever (CCHF) virus was investigated individually in tick species known to be vectors or carriers of this virus. In almost half of the tick pools (48%), DNA from at least one pathogen was detected and eleven species of TBPs from six genera were reported. TBPs were found in ticks from all collected hosts and were present in more than 80% of the investigated area. The detection of DNA of certain species confirmed the previous identification of these pathogens in Corsica, such as Rickettsia aeschlimannii (23% of pools), Rickettsia slovaca (5%), Anaplasma marginale (4%) and Theileria equi (0.4%), but most TBP DNA identified had not previously been reported in Corsican ticks. This included Anaplasma phagocytophilum (16%), Rickettsia helvetica (1%), Borrelia afzelii (0.7%), Borrelia miyamotoi (1%), Bartonella henselae (2%), Babesia bigemina (2%) and Babesia ovis (0.5%). The high tick infection rate and the diversity of TBPs reported in this study highlight the probable role of