Culicoides biting midges are small hematophagous flies, some species of which transmit pathogens to livestock and humans. In the Brazilian Amazon Basin, Culicoides diversity has remained largely unexamined. Jamari National Forest (JNF) is a conservation area in Rondônia State where research and environmental education are conducted in conjunction with the protection of natural resources, and the recovery of degraded areas.The present study augments our knowledge of Culicoides diversity in JNF. The survey collected 640 individuals from 41 species; 632 individuals were collected by HP light trap and 8 were collected by the BG Sentinel trap. Twelve species are first-time records for Rondônia: Culicoides acotylus Lutz, Culicoides albuquerquei Wirth & Blanton, Culicoides baniwa Felippe-Bauer, Culicoides bricenoi Ortiz, Culicoides brownei Spinelli, Culicoides carsiomelas Wirth & Blanton, Culicoides debilipalpis Lutz, Culicoides forattinii Ortiz, Culicoides ginesi Ortiz, Culicoides profundus Santarém, Felippe-Bauer & Trindade, Culicoides pseudoreticulatus Santarém, Felippe-Bauer & Castellón, and Culicoides spurius Santarém, Felippe-Bauer & Trindade. The most abundant species were Culicoides glabrior Macfie, Culicoides pseudodiabolicus Fox, Culicoides debilipalpis, Culicoides quasiparaensis Clastrier, and Culicoides brownei Spinelli. The present study updates the Culicoides species count in Rondônia to 55, and highlights the importance of JNF as a hotbed for biting midge diversity.