About 1000 freshwater fishes have been found so far in the Orinoco River Basin of Venezuela and Colombia. This high ichthyological diversity reflects the wide range of landscapes and aquatic ecosystems included in the basin. Mountain streams descend from the high Andes to become rapid‐flowing foothill rivers that burst out upon vast savannah flatlands where they slowly make their way to the sea. These white‐water rivers are heavily laden with sediments from the geologically young Andes. Because their sediment deposits have formed the richest soils of the basin, they have attracted the highest density of human populations, along with the greatest levels of deforestation, wildfires, agricultural biocides and fertilizers, sewage and all the other impacts associated with urban centres, agriculture and cattle ranching. In the southern portion of the basin, human populations are much smaller, where often the only inhabitants are indigenous peoples. The ancient rocks and sands of the Guiana Shield yield clear and black water streams of very different quality. Here, sediment loads are miniscule, pH is very acid and fish biomass is only a fraction of that observed in the rich Andean tributaries to the north. For each region of the basin, the current state of knowledge about fish diversity is assessed, fish sampling density evaluated, the presence of endemic species and economically important species (for human consumption or ornamental purposes) described and gaps in knowledge are pointed out. Current trends in the fishery for human consumption are analysed, noting that stocks of many species are in steep decline, and that current fishing practices are not sustainable. Finally, the major impacts and threats faced by the fishes and aquatic ecosystems of the Orinoco River Basin are summarized, and the creation of bi‐national commissions to promote standardized fishing laws in both countries is recommended.