The freshwater molluscan assemblage from the uppermost Eocene Florissant Formation (34.07 ± 0.10 Ma), Colorado, USA, provides a reliable proxy in reconstructing past ecology and environmental characteristics of ancient Lake Florissant. In particular, stable-isotope analyses of aragonitic shell material contribute to our understanding of the paleohydrologic history of this ancient lake. Re-examination of molluscan taxonomy in the middle shale and caprock conglomerate (informal) units produces three sphaeriid bivalves (family Sphaeriidae, genus Sphaerium) and two pulmonate gastropod genera (family Planorbidae, genus Gyraulus; and family Lymnaeidae, genus Lymnaea). The middle-shale assemblage, representing quiet-lake deposition, is dominated by pulmonate gastropods; the shell material in all specimens in this unit has been replaced by silica. The caprock conglomerate assemblage, representing redeposition by a debris flow, is dominated by bivalves; specimens within the caprock conglomerate unit are aragonite, interpreted to be biogenic (original). Carbon and oxygen isotope analyses of the aragonite show a strong covariance (all molluscan data: r = 0.83; sphaeriids: r = 0.76) with consistent grouping (δ13C: 0‰ to −5.5‰, δ18O: +2‰ to −5‰) from all families. This result indicates that ancient Lake Florissant was “closed” and that evaporation had a stronger effect on isotopic values than precipitation, for the duration of the sampled interval. This finding is in agreement with our current understanding of how the sediments of the Florissant Formation were deposited within a lake that formed because of a paleoriver being dammed by debris flows.