The Napo River, a tributary of the Amazon in northeastern Ecuador, harbors a high diversity of fish and macroinvertebrates, with communities varying according to altitude and ecosystem type. Its tributary, the Curaray River, is one of the last free-flowing rivers in Ecuador, yet its aquatic fauna remains largely undocumented. This study presents the most comprehensive list of fish and macroinvertebrates for this basin and evaluates how altitude influences species diversity and composition. A total of 28 sites were sampled across three altitudinal zones (514, 285, and 209 m), covering streams, lagoons, and rivers during the dry season of 2018. For fish, the literature reports 331 species across 90 families and 13 orders, 69% of which were collected in this study. For macroinvertebrates, no prior studies exist for the Curaray, and 102 genera were recorded in the present study. Linear models were applied to analyze the relationship between altitude and α-diversity, along with community composition or β-diversity analyses (Jaccard dissimilarity index, PERMANOVA, NMDS, and INDVAL). Results show that α-diversity of both fish and macroinvertebrates decreases with altitude, though different patterns emerge in intermediate zones. Fish community composition varied significantly across altitudes and habitats, whereas macroinvertebrate composition changed only with altitude. These findings align with previous Amazonian studies and highlight the importance of lowland ecosystems as critical hotspots for aquatic biodiversity, as they provide more favorable conditions for fish diversity, such as warmer temperatures, greater habitat heterogeneity, and higher primary productivity. The high diversity recorded underscores the need for conservation, particularly in regions like the Curaray, where environmental conditions remain largely undisturbed.