Knowledge of the fish assemblages of Andean mountain streams is restricted to accessible areas. This study is the first biological survey of the remote Suaza River in the Colombian Andes, at its source in the Cueva de los Guácharos National Park. Mechanistic insight into the ecological patterns and gradients of the river is provided, focusing on the spatial distribution of fish communities in relation to environmental variables and habitat characteristics. Electrofishing surveys and macroinvertebrate sampling were carried out, the river habitat was characterized, three environmental quality indices were applied, and potential water pollutant concentrations were measured. Twenty-seven fish species were recorded — two of them exotic (Poecilia reticulata and Xiphophorus hellerii) — as well as 45 macroinvertebrate families. Fish species richness was higher at lower altitudes with gentle slopes, where habitat temperature, size and heterogeneity were higher. Although macroinvertebrate diversity slightly increased downstream following a similar pattern as fish species richness, it declined at lower sites. Fish assemblages were distributed in three clusters corresponding to headwaters, middle sections and lower reaches of the river, showing correlations with environmental factors, suggesting habitat preferences. Although the headwaters of the Suaza River remains pristine inside the park, human influence is responsible for ecosystem alteration downstream.