Successful conservation strategies require consideration of species interactions. In their life cycle, freshwater mussels of the family Unionidae depend on a suitable host fish on which their larvae metamorphose into juveniles. This study investigated the host–parasite interaction of different fish species with the endangered European thick-shelled river mussel (Unio crassus) under artificial and natural conditions. Chondrostoma nasus, Cottus gobio, Leuciscus idus, Phoxinus phoxinus, Squalius cephalus, Scardinius erythrophthalmus and three different strains of Gasterosteus aculeatus were identified as suitable hosts. In contrast, U. crassus was not able to metamorphose on Acipenser ruthenus and Rutilus rutilus. In natural U. crassus streams, 16 different fish species were found, with pronounced differences in the occurrence and abundance of suitable hosts. Data from the laboratory infections and from the field investigations strongly suggest that S. cephalus is the most important host for U. crassus in the Danube drainage, despite the fact that some populations exclusively depend on other hosts. The results of this study indicate that (i) an evaluation of host fish importance in unionid mussels should not exclusively rely on standardized infestation experiments without considering natural fish communities, (ii) host suitability can vary within different strains of the same host species, (iii) development time of U. crassus on different hosts varies considerably, and (iv) natural recruitment in functional U. crassus populations can be secured by highly different fish community structures and densities. The wide host range of U. crassus is likely to increase the chances of successful restoration of priority populations for conservation.