Identifying fish spawning areas is of great ecological and conservation importance as fishes are suffering increasing threat levels from anthropogenic activities. However, to date very few studies have done so in the Amazon basin. In the Beni River located in the upper Madeira basin, fishers reported that a particular ecotone near the town of Rurrenabaque was a fish reproduction area. To test the importance of this zone as a spawning site, we conducted an ichthyoplankton survey during the month when reproduction is most likely to occur. The specific identification of larvae and eggs was made with a metabarcoding analysis. With this approach 13 different fish species of high importance for regional and local fisheries were identified, including the long-distance migratory gilded catfish (Brachyplatystoma rousseauxii), considered as endangered in the upper Madeira. Combining the development time of morula-gastrula egg stages with the integrated current velocity of the river, we identified a spawning area that ranges between the last gravel beaches close to Altamarani community and the San Miguel del Bala community on top of Suse strait, the last foothill of the Andes. This spawning area further extends upstream in the Madidi and Pilon Lajas protected areas and Tacana and T’simane indigenous communities, when considering eggs in final embryo stage. This portion of the Beni River, at the Andean foothills, is heavily impacted by anthropogenic activities, ranging from unmanaged fisheries to contamination coming from the extraction of fluvial aggregates, upstream gold mining, deforestation associated with a sugar cane mill, an existing downstream dam, and projected upstream dam projects. Some urgent solutions for the conservation of this already impacted area are proposed. Confirming fish spawning zones, although difficult, is crucial to inform the definition of priority areas for conservation and management measures, in particular when these sites host endangered species.