Habitat alteration is one of the major drivers of species loss. Springs may be among the least affected aquatic habitats and are considered to be refugial habitats. Diatom assemblages were sampled from 74 Swiss springs comprising seven spring types over a wide altitudinal and ecological range as well as a wide range of anthropogenic modifications (alterations). Each spring was assigned to a five-point alteration scale, from natural to heavily altered. In total, 504 diatom species were recorded (not including 36 taxa that could not be identified to species level) from five microhabitats, and coded according to rarity and Red List status. The results of this extensive spring-habitat diatom survey were compared statistically with a large stream diatom dataset (DI-CH). The spring diatom microflora was more species rich and included more rare and/or threatened species than the stream microflora. The proportion of Red List species and rare species was highest in the DI-CH dataset, but the proportion of species with no Red List status and rare species was the highest in springs. Species richness, Red List status, and rarity were significantly related to the degree of spring alteration. This is consistent with the hypothesis that unaltered springs function as refuges for the Swiss fresh water microflora, and that they can be regarded as “least-impaired habitats”. These may be critically important for the conservation of the European freshwater diatom microflora.