Fish assemblages inhabiting the mountain streams of the Guiana shield have scarcely been studied. Here we investigated fish assemblages in eight headwater mountain streams of the Mount Itoupé (French Guiana), and tested how local freshwater fish assemblages are shaped by environmental characteristics. We show that Mount Itoupé streams host uneven species assemblages, characterized by low species richness (less than ten species per site) and a high proportion of small Siluriform species. Differences in fish assemblage composition between the sites were mainly due to basin identity, but regional climate and position of the site in the river gradient also had a significant effect, although of lower magnitude. In contrast, local habitat hardly affected fish richness, abundances or assemblage composition. Species richness increased along the upstream–downstream gradient, whereas fish abundance depended mainly on the slope orientation of Mount Itoupé. Although these results need confirmation on a larger amount of sampling sites, located on other Guianese mountains, this preliminary study shows that the mountain streams of the Guiana Shield, although rarely investigated, host not only uneven species assemblages, but also present an original combination of environmental determinants shaping fish assemblage structure. Those rare and original ecosystems hence deserve more attention and should be preserved from human disturbances.