Accelerated glacier melt and runoff may lead to inputs of labile dissolved organic matter (DOM) to downstream ecosystems and stimulate the associated biogeochemical processes. However, still little is known about glacial DOM composition and its downstream processing before entering the ocean, although the function of DOM in food webs and ecosystems largely depends on its composition. Here, we employ a set of molecular and optical techniques (UV–vis absorption and fluorescence spectroscopy, 1H NMR, and ultrahigh-resolution mass spectrometry) to elucidate the composition of DOM in Antarctic glacial streams and its downstream change. Glacial DOM consisted largely of a mixture of small microbial-derived biomolecules. 1H NMR analysis of bulk water revealed that these small molecules were processed downstream into more complex, structurally unrecognizable molecules. The extent of processing varied between streams. By applying multivariate statistical (compositional data) analysis of the DOM molecular data, we identified molecular compounds that were tightly associated and moved in parallel in the glacial streams. Lakes in the middle of the flow paths enhanced water residence time and allowed for both more DOM processing and production. In conclusion, downstream processing of glacial DOM is substantial in Antarctica and affects the amounts of biologically labile substrates that enter the ocean.