Human land-use is changing Earth’s surface, causing a decline in biodiversity and altering ecosystem functioning. However, most of the empirical evidence of land-use impacts in the Neotropics comes from studies investigating isolated land-use types, and the pathways by which intensified land-uses affect ecosystem functioning are largely unknown. Using a database from 61 streams spanning two hyperdiverse Neotropical regions, we demonstrate that intensive human land-uses (agriculture, urbanization, pasture, and afforestation) strongly affect stream biodiversity and functioning. We showed negative associations of agriculture, pasture and urbanization with taxonomic richness, functional diversity, and diversity of trait categories (recruitment and life-history, resource and habitat-use, and body size) of fish, arthropod, and macrophyte. The impacts of intensive land-uses on standing biomass were negative and driven by direct and indirect effects mediated by declines in taxonomic and functional diversities. Our findings highlight that human land use can reshape stream biodiversity, with multiple negative consequences on ecosystem functioning.