This study investigated and compared the diatom flora from thermo-mineral springs in Auvergne (France) and Sardinia (Italy). Samples were collected from rock/cobbles and fine sediments in 16 springs between January 2015 and March 2017. A total of 207 taxa (59 genera) were found. Multivariate analyses revealed significant differences in species composition and abundance among diatom assemblages both within each geographic region and between Auvergne and Sardinia (global R = 0.516; p = 0.002), suggesting the importance of local and climatic factors in species distribution. Based on abundance and common occurrence in multiple sites, some taxa can be considered more representative of springs in each region: Crenotia thermalis for Auvergne and Lemnicola exigua, Nitzschia amphibia, N. inconspicua and Rhopalodia operculata for Sardinia. pH, conductivity and HCO3- were the most significant environmental variables for diatom assemblages. Our results highlight the high heterogeneity of these spring systems. Future taxonomic insights can be useful to define the identity of some abundant and dominant taxa not identified at the species level in this study. Their identification is a crucial step for a more precise ecological characterization and comparison of these peculiar spring systems.