This study examines the Holocene history of Río Seco Lake (3040 m a.s.l; Sierra Nevada, Southern Spain) by analysing diatom remains and other paleoenvironmental data. The aim is to understand the impact of long-term environmental and climatic variability on the aquatic ecosystem over the past 21,000 years. Our results suggest that shifts in diatom assemblages were mainly climate-driven in terms of temperature and water availability. The absence of diatom remains during the Late Pleistocene indicated low temperatures and prolonged lake snow cover. Five distinct periods were identified during the Holocene. The high abundance of epiphytic and bog-inhabiting taxa and tychoplanktonic Tabellaria flocculosa in the period 11,000–6700 cal yr BP were indicative of a humid climate. The onset of the tychoplanktonic Aulacoseira alpigena between 6700 and 5100 cal yr BP indicated a drop in temperature. These changes intensified during the period 5100–3300 cal yr BP, when the most significant changes in diatom assemblages took place with the dominance of A. alpigena and an abrupt increase in the abundance of the epiphytic Fragilaria radians. During the subsequent period (3300–1500 cal yr BP), the significant declines in A. alpigena and in epiphytic taxa were indicative of increased aridity and higher alkalinity values due to increased aridity and Saharan dust input during this period. The last period (1500–256 cal yr BP) was characterized by a rise in the abundance of Staurosirella pinnata, indicative of warmer temperatures and higher alkalinity values coincident with a marked increase in proxies of temperature and aridity. The increase in aridity and temperature during the last period, which has led to changes in diatom assemblages, is a matter of great concern in an ecosystem that is particularly susceptible to global warming.