The conservation of endangered affiliate species, which are critically dependent on the presence of another species, is often hindered by a poor understanding of the relationships between the interacting partners. The parasitic stage of endangered unionid bivalves constitutes a tight host–affiliate linkage between the mussels and their host fishes. However, the threats resulting from potential shortages of the host species are rarely sufficiently quantified and incorporated into conservation strategies. In this paper, we integrated both host quality and availability analyses to assess the potential threats to the endangered thick-shelled river mussel Unio crassus that result from the impairment of its host resources in Central Europe. The experimental determination of the compatibility of U. crassus parasitic larvae (glochidia) with its potential host fishes revealed an intermediate level of host specificity. At least, some glochidia successfully developed on 14 of the 27 potential hosts that were evaluated. Nevertheless, only three fish species (Scardinius erythrophthalmus, Phoxinus phoxinus and Cottus gobio) enabled the majority of the attached glochidia to transform successfully. Subsequently, our analysis of host availability at sites inhabited by living or extirpated populations of U. crassus showed that the local extirpations of U. crassus are likely associated with an impaired status of the fish assemblage and with the absence of the primary host fishes. These results indicate that the availability of host fish resources may have played at least an additive role in the present pan-European decline of U. crassus and that the evaluation of host limitation without precise data on the host compatibility or host abundance may be ineffective for identifying the threats to particular species. It demonstrates a strong need for more thorough incorporation of host limitation issues into conservation strategies for U. crassus and probably also for other species of freshwater mussels that were previously considered safe from host limitation.