Indonesian freshwater fish diversity is threatened by human activities such as logging, land clearing, pollution and introduction of non-native species. The latter may pose serious threats to endemic freshwater fauna even in relatively pristine and isolated habitats. One such area, West Papua in the island of New Guinea, is one of the least studied regions in the world and a biodiversity hotspot. The Mamberamo River contains the highest proportion of non-native fish compared to other major river systems in New Guinea. To document this, we conducted a field study to validate and further temporally characterise the fish biodiversity to ascertain its current status. Since the last ichthyological survey 15 years ago, we detected two additional non-native species (Leptobarbus melanopterus and Oreochromis niloticus) that have established in the river system. Moreover, our survey revealed that non-native fish are extremely common in the midreaches of the Mamberamo River, comprising 74% of total catch, with non-native Barbonymus gonionotus (family Cyprinidae) now established as the dominant species. The biomass of non-native B. gonionotus now exceeds that of all native fish combined in the main river channel. These results highlight the serious threat of invasive species in remote regions that support high levels of endemic biodiversity. Plans for containment, prevention through education programmes, and management are urgently required.