Colombia is one of the hotspots regarding mosquito biodiversity and mosquito-transmitted diseases. Endemic, tropical diseases like malaria and dengue are major public health issues and novel viruses of zoonotic origin like Chikungunya and Zika are emerging. Nevertheless, little is known about the mosquito vector species circulating in remote Colombian regions and the mosquito-associated viromes. In a pilot study carried out from August 2014 to December 2016, more than 8000 mosquitoes (Diptera: Culicidae) were collected in the Atrato River valley in Northwest Colombia to identify the mosquito species in the region and get information on the species abundance, biting behaviour, and phylogeny of these mosquito species. In addition, a PCR- and NGS-based virus search was promoted in the most abundantly collected mosquito species to get insight into the virome of each mosquito species and identify yet undescribed mosquito-associated viruses, sequence their genomes, establish their phylogenetic relationships with formerly described viruses and describe their genome organizations. More than 43 mosquito species of 14 genera could be identified by morphology and molecular markers (Cytochrome c oxidase subunit 1, internal transcribed spacer 2), and their biting behaviours and phylogenetic relationships were described. Mansonia titillans (17,7%), Psorophora albipes (16,5%), Anopheles darlingi (15,8%), Anopheles nuneztovari (10,9%), Coquillettidiae venezuelensis (8,9%), and Coquillettidiae lynchi (8,6%) were the most abundantly collected mosquito species, each bearing a species-specific virome. Moreover, 46 human blood samples from patients with suspicion of malaria were screened for viruses. A total of 64 novel viruses were discovered in mosquitoes and human blood samples. The genomes of these viruses were sequenced and annotated, and the viruses were assigned to a taxonomic rank by phylogenetic analyses. Each of the mosquito species just mentioned has been involved in the transmission of zoonotic diseases. The Atrato River valley thus harbours a high diversity of mosquito species and associated undiscovered viruses, the majority being insect and plant viruses. Systematic monitoring of mosquito species diversity and mosquito-associated viruses in biodiversity hotspots could be a promising approach for the prevention and control of novel zoonotic diseases.