AbstractSulfate is increasingly found in elevated concentrations in freshwater ecosystems due to anthropogenic activities. Chronic exposure to sulfate has been reported to cause sublethal effects on freshwater invertebrates. Previous sulfate toxicity tests have mostly been conducted in hard or moderately hard waters, and research on species inhabiting soft water is needed, given that freshwater organisms face heightened sensitivity to toxicants in water of lower hardness. In the present study, we examined sulfate sensitivity of two endangered freshwater mussel species, Unio crassus, and Margaritifera margaritifera. Glochidia and juveniles of both species were subjected to acute and/or chronic sulfate exposures in soft water to compare sulfate sensitivity across age groups, and effective concentrations (EC)/lethal concentrations (LC) values were estimated. Mussels were individually exposed to allow relatively larger numbers of replicates per treatment. Chronic sulfate exposure significantly reduced growth, foot movement, and relative water content (RWC) in juvenile mussels of M. margaritifera. Mussels at younger stages were not necessarily more sensitive to sulfate. In the acute tests, LC50 of glochidia of M. margaritifera and U. crassus was 1301 and 857 mg/L, respectively. Chronic LC10 was 843 mg/L for 3-week-old U. crassus juveniles, 1051 mg/L for 7-week-old M. margaritifera juveniles, and 683 mg/L for 2-year-old M. margaritifera juveniles. True chronic Lowest Effective Concentration for 7-week-old M. margaritifera may be within the 95% interval of EC10 based on RWC (EC10 = 446 mg/L, 95%CI = 265–626 mg/L). Our study contributed to the understanding of sulfate toxicity to endangered freshwater mussel species in soft water.