Over the past century, the abundance of many plant species has declined noticeably, including in the herbaceous understory of temperate deciduous forests. Two separate censuses of Trelease Woods in Illinois, USA, one in 1994–95 that was repeated in 2015, and a second made annually from 1994–2015, indicated major losses for herb species. Quadrat frequency, flowering quadrat frequency, number of species, number of species in flower, number of individuals, and species diversity (H′) all declined greatly over the census period. Most species saw a marked decline in frequency and density, with the number of individuals declining by two-thirds and some species disappearing entirely. Gains from new colonization were minor in comparison. Among persisting species, almost all became shorter and stopped flowering. Number of individuals per species in 1994 and percentage of individuals lost by 2015 were not related. Spring ephemeral species had less loss in frequency and percentage of individuals lost per species than four other phenological syndromes. Although multiple factors changed during the study period, the specific mechanisms behind these biodiversity changes were not addressed by the study. This biodiversity loss is expected to continue and persist with little chance of recovery for dispersal-limited herb species in this isolated, fragmented forest.