Abstract
<title>Abstract</title> <p> <italic>Culicoides</italic> Latreille biting midges (Diptera: Ceratopogonidae) are hematophagous insects of medical and veterinary importance due to their role in arbovirus transmission. In the neotropical region, species diversity remains incompletely characterized, particularly in the Amazon Basin. This study assessed the diversity and composition of <italic>Culicoides</italic> in Rondônia, Brazil, compared assemblages between forest fragments and peridomiciliary environments, identified vertebrate blood meal sources, and evaluated the presence of Oropouche virus (OROV) RNA. Specimens were collected using light traps and protected human attraction and identified based on morphological characters. Blood meals were analyzed using mitochondrial DNA markers, and RT-qPCR performed OROV detection. A total of 1,058 specimens were examined, representing 76 taxa, including 46 identified species and 30 morphotypes. The most abundant species were <italic>Culicoides leopoldoi</italic> Ortíz, <italic>Culicoides glabellus</italic> Wirth & Blanton, and <italic>Culicoides pseudodiabolicus</italic> Fox. Nine species were recorded for the first time in Rondônia, including four new records for Brazil. Forest fragments exhibited higher species richness and diversity than peridomiciliary areas, which harbored fewer species but included taxa of epidemiological relevance. Blood meal analysis revealed a predominance of avian hosts, particularly <italic>Gallus gallus</italic> Linnaeus, as well as mammals, including humans, indicating opportunistic feeding behavior. No OROV RNA was detected in the 414 pools analyzed. These findings demonstrate high <italic>Culicoides</italic> diversity in western Amazonia and highlight the concentration of potential vector species at forest–human interfaces. Although no evidence of OROV circulation was detected during the sampling period, the presence of anthropophilic and generalist species underscores the importance of continued surveillance. </p>