Arctic river watersheds are important components of the global climate system and show an amplified response to climate change. Here, we characterize origins and transformations of dissolved organic matter (DOM) in five major Arctic rivers (Kolyma, Lena, Yenisei, Ob, Mackenzie) over 3 years with seasonal sampling periods using measurements of carbohydrates, amino acids, bacterial biomarkers (D-amino acids), and plant protein biomarkers (hydroxyproline). A strong seasonal cycle of bioavailable DOM export was observed that correlated with discharge, vegetation, river morphology and water residence time. The chemical composition of bioavailable DOM was different among rivers reflecting unique characteristics of Arctic river watersheds. Trends in specific bacterial biomarkers were synchronous to changes in bacterial community compositions demonstrating that bacterial communities responded to the seasonal shifts in organic matter quality and chemical composition. Extensive heterotrophic processing of plant and soil-derived DOM resulted in major inputs of bacterial detritus, and bacterial organic matter accounted for 21–42% of DOC in all watersheds. Dissolved organic nitrogen sources were dominated by bacterially-derived nitrogen and important contributions of soluble plant protein during the Spring freshet. Overall, our results demonstrated the importance of watershed characteristics and bacterial metabolism in regulating DOM composition, reactivity and carbon fluxes in Arctic river watersheds.