Alpine lakes are well known as “environmental sentinels”, which are sensitive to anthropogenic impacts, and paleoecological studies provide useful information regarding the effects of local and global changes. In the present study we investigate, for the first time, the temporal variations in testate amoeba assemblages in relation to environmental factors and anthropogenic impacts in a high-altitude lake located in the Italian Alps (Balma Lake, Piedmont, Italy). The testate amoebae are well-suited as bioindicators and have been employed to reconstruct past climate and environmental changes from lake sediments. However, these organisms have not been thoroughly studied in alpine lakes, especially in Europe. We detected significant changes in testate amoeba assemblages along an alpine lake sedimentary profile over the last 2000 years. These changes were mainly associated with variations of trace elements, nutrients, and grain size. Moreover, five different time periods were identified and related to pluvial/drought events of the last 2000 years and to the “Late Antique Little Ice Age” (LALIA). Alpine lakes are originally fishless and fish introductions for recreational purposes could cause important changes in these ecosystems. However, the introduction of brook trout in Balma Lake during the 1970s seems to have had little influence on testate amoeba assemblages. The present work provides new insights for the evaluation of temporal changes in testate amoebae through paleoecological investigations. Similarly, the comparison of testate amoeba and chironomid proxies offered an important tool for analyzing recent climatic and environmental changes in alpine lakes (in lacustrine environments), especially for the European Alps.