Loss of biodiversity within relatively pristine protected areas presents a major challenge for conservation. At La Selva Biological Station in the lowlands of Costa Rica, amphibians, reptiles, and understory birds have all declined over the past four decades, yet the factors contributing to these declines remain unclear. Here, we conduct two tests of the hypothesis that faunal declines are linked to shifting dynamics of leaf litter, a critical microhabitat for amphibians and reptiles and a major component of forest carbon cycles. First, we conduct a 16-month manipulation of leaf litter and measure response by terrestrial amphibians and reptiles. Second, we synthesize three year-long datasets collected over four decades to evaluate potential multi-decade change in standing litter depth. We show that litter depth regulates density of amphibians and reptiles, and that the strongest response to manipulations is in species that decline most rapidly based on long-term data. Our synthesis of litter depth data suggests considerable interannual variability in standing stocks of leaf litter with lowest quantity of leaf litter in the most recent sampling period. These tests are consistent with the hypothesis that these faunal declines may be in part driven by changes in forest litter dynamics, and ultimately to climate-sensitive carbon cycles.