Rivers act as dynamic connectors between terrestrial and marine ecosystems, transporting and transforming materials along the aquatic continuum. We investigated longitudinal biogeochemical patterns across the Rapel catchment (central Chile) by integrating dissolved organic matter (DOM) characterization, dissolved metals and nutrients, and microbial community profiling from the Andean headwaters to the Pacific Ocean. Headwaters were characterized by low DOC and nutrient concentrations, high protein-like fluorescence, and diverse microbial assemblages dominated by Flavobacterium, Polaromonas, and Rhodoferax. Downstream, agricultural and mining activities increased nutrient and metal concentrations and altered microbial community composition with an increasing prevalence of hgcI_clade. The Rapel Reservoir emerged as a major biogeochemical discontinuity, promoting DOM transformation, restructuring microbial assemblages, and modifying downstream chemical conditions. Overall, tributary inputs, land use, and hydrological regulation disrupted simple longitudinal patterns and generated distinct biogeochemical processing zones. These findings highlight the interplay between downstream transport and local processing in shaping riverine functioning in Mediterranean Andean catchments and provide a basis for assessing future climate and land-use impacts on riverine biogeochemistry.