Tropical high-altitude lakes are vital freshwater reservoirs in the Andean regions. They are heavily threatened by human activities that may alter their functioning and hamper the provisioning of key ecosystem services such as water supply. Despite their ecological and social relevance, we know little about these waterbodies, especially regarding the factors influencing their functioning. Here, we explored the links between several environmental variables and phytoplankton productivity, measured as chlorophyll-a concentration and total phytoplankton biovolume. For this, we sampled twenty-four tropical high-altitude lakes located over three-thousand meters above sea level in Southern Ecuador. We found that four abiotic factors combined explained 76% of the variation in chlorophyll-a concentration amongst lakes. Contrary to what studies from temperate regions suggest, taxa richness was not related to either chlorophyll-a concentrations or total phytoplankton biovolume. Moreover, phytoplankton biovolume diversity was negatively correlated to both chlorophyll-a concentrations and total phytoplankton biovolume. This was due to a very uneven distribution of productivity amongst taxa in the more productive lakes. To the best of our knowledge, this is the first attempt to explore the determinants of phytoplankton functioning in tropical high-altitude lakes. We hope that this study will help to establish a baseline for evaluating the consequences of human activities in the ecology and functioning of this vital but fragile ecosystem. Our results suggest that by modifying the abiotic and biotic parameters of tropical high-altitude lakes, human activities can indirectly impact their functioning and their capacity to provide vital ecosystem services.