Unionid mussels are among the most threatened groups of organisms globally. Habitat loss is one of the major drivers of their declines and local extinctions. In Poland six native species occur, but data on their present distribution are limited. The aim of this study was to assess unionid mussel species composition and distribution in the Pisa River, protected as part of Natura 2000 areas but currently threatened by plans of modification of its discharge regime and transformation into a navigable waterway. Twenty-one sites were surveyed using the timed-search method. The strictly protected Unio crassus was present at 86% of the sites and showed continuous distribution throughout the river. Young mussels constituted 26% of U. crassus individuals, indicating successful recruitment. Pseudanodonta complanata was found at three sites, suggesting that the Pisa River provides an important habitat also for this endangered species. Additionally, Anodonta cygnea, A. anatina, U. pictorum, and U. tumidus were recorded. Such multispecies unionid mussel communities are increasingly rare and need to be protected. The Pisa River is characterised by natural flow regime and a meandering channel with diversified depth and flow speeds. Conservation of U. crassus and other unionid mussels requires that its natural character is maintained and no changes are introduced into its channel.