Uruguayan Southern lagoons exhibit high Holocene resolution paleoenvironmental-paleoclimatic records for inferring long-term regional changes. The multiproxy analysis of three sediment cores enabled the recognition of Holocene climatic variability from the paleolimnological record of the Peña lagoon over the last 2,458 cal. years BP. Four main stages were identified throughout the record. The first stage (2,458–1,500 cal. years BP) was characterized as a shallow meso-eutrophic system with high abundances of aerophilic benthic species, epiphytic taxa and planktonic taxa. The second stage (1,415–390 cal. years BP) showed a noticeable change in the diatom assemblage dominated by the fresh-brackish benthic species Staurosira construens, but also presented fluctuations in the abundance of Aulacoseira ambigua and Aulacoseira granulata, which indicate the occurrence of temperate to cold and semiarid climatic conditions including intervals of high rainfall. The core chronology allowed us to ascribe this stage to the Little Ice Age (LIA). The third stage, post 390 cal. years BP, showed the highest proportion of freshwater planktonic species throughout the entire core, thus indicating the development of an eutrophic system under relatively warm and wet conditions, which were assigned to the Current Warm Period. After ca. 1,962 AD, a sharp increase in the abundance of epiphytic species highlights the onset of the fourth stage, which was characterized by littoral expansion and, consequently, the proliferation of associated macrophytes due to anthropogenic impacts.