Biodiversity loss is a major global environmental challenge, driven primarily by human activities like land-use change and climate change. While tropical ecosystems were once thought to be relatively stable, emerging evidence shows significant declines even within protected tropical forests. Here, we used a 27-year monitoring program to: (1) quantify long-term changes in the richness and composition of aquatic insect assemblages in two tropical headwater streams in Costa Rica, and (2) evaluate how shifts in regional climate, particularly precipitation patterns, might be driving these changes. We hypothesized that taxonomic richness has been declining and that these declines are associated with a new disturbance regime due to changes in precipitation. Aquatic insects were sampled from 1997 to 2024 in two headwater streams at La Selva Research Station, Costa Rica. Samples were collected with a corer in run habitats by the same technician over the entire period. Insects were identified, mostly to genus level, and taxonomic richness and assemblage composition were assessed. Stream discharge, precipitation, and air temperature data were collected as potential environmental drivers. We found 87 and 96 taxa in each stream, with Diptera being the most diverse and abundant order. There was a significant exponential decline in richness in both streams, dropping to about one-third of the original value by 2016, before showing a minor increase in the 2024 sampling event. The density of nearly all dominant groups also declined by up to 90%. The assemblage composition became progressively more dissimilar over time, reaching maximum difference by 2016, although the 2024 assemblage showed a partial overlap with earlier years. Our 27-year study reveals a concerning and robust pattern of biodiversity loss in protected tropical headwater streams, with a consistent decline in richness and abundance across almost all taxa. While no taxa showed significant increases, the partial recovery observed in 2024 suggests that some resilience may exist. The observed declines and resulting changes in community composition are linked to climate change-related factors and long-term hydrological variability. The findings underscore that protected tropical forests are undergoing pervasive, multifaceted biodiversity loss, highlighting an urgent need for long-term research to identify ecosystem consequences.