Background: The south-west insular territories of the Indian Ocean have recently received attention concerning the diversity of arthropods of medical or veterinary interest. While a recent study highlighted the circulation of Culicoidesborne viruses, namely bluetongue and epizootic hemorrhagic disease, with clinical cases in Mayotte (comprising two islands, Petite-Terre and Grand-Terre), Comoros Archipelago, no data have been published concerning the species diversity of Culicoides present on the two islands. Results: A total of 194,734 biting midges were collected in 18 sites, covering two collection sessions (April and June) in Mayotte. Our study reports for the first time livestock-associated Culicoides species and recorded at least 17 described Afrotropical species and one undescribed species. The most abundant species during the April collection session were C. trifasciellus (84.1%), C. bolitinos (5.4%), C. enderleini (3.9%), C. leucostictus (3.3%) and C. rhizophorensis (2.1%). All other species including C. imicola represented less than 1% of the total collection. Abundance ranged between 126–78,842 females with a mean and median abundance of 14,338 and 5111 individuals/night/site, respectively. During the June collection, the abundance per night was low, ranging between 6–475 individuals. Despite low abundance, C. trifasciellus and C. bolitinos were still the most abundant species. Culicoides sp. #50 is recorded for the first time outside South Africa. Conclusions: Our study reports for the first time the Culicoides species list for Mayotte, Comoros Archipelago, Indian Ocean. The low abundance and rare occurrence of C. imicola, which is usually considered the most abundant species in the Afrotropical region, is unexpected. The most abundant and frequent species is C. trifasciellus, which is not considered as a vector species so far, but its role needs further investigation. Further work is needed to describe Culicoides sp. #50 and to carry on faunistic investigations on the other islands of the archipelago as well as in neighboring countries.