The Ecuadorian portion of the Marañón drainage remains poorly explored, and its ichthyofauna is largely undocumented. Here, we present the first annotated checklist of fish species from the Conambo River, a major tributary of the Tigre River, together with an analysis of diversity patterns. A total of 108 species representing seven orders were recorded; however, the species-accumulation curves did not reach an asymptote, suggesting that the true richness is likely higher. Fish community composition varied markedly across seasons and along the river’s longitudinal gradient, reflecting high beta diversity. The Conambo River also sustains local livelihoods: 17 species are regularly consumed by the Shiwiar people and 33 by the Zápara. Likewise, several commercially valuable taxa: Pseudoplatystoma punctifer, Prochilodus nigricans, and Mylossoma albiscopum, use it as a refuge from intense fishing pressure elsewhere. Notably, we documented an exceptional diversity of Loricariidae rarely observed in other basins. The river’s high species richness, its cultural significance for the Shiwiar and Zápara peoples, and the occurrence of Key Biodiversity Area species, such as Bujurquina pardus and Melanocharacidium rex, underscore the urgent need to designate the Conambo as a protected area.