The impact of human activities on the concentrations and composition of dissolved organic matter (DOM) and particulate organic matter (POM) was investigated in the Walloon Region of the Meuse River basin (Belgium). Water samples were collected at different hydrological periods along a gradient of human disturbance (50 sampling sites ranging from 8.0 to 20,407 km2) and during a 1.5 year monitoring of the Meuse River at the city of Liège. This dataset was completed by the characterization of the DOM pool in groundwaters. The composition of DOM and POM was investigated through elemental (C:N ratios), isotopic (δ13C) and optical measurements including excitation emission matrix fluorescence with parallel factor analysis (EEM–PARAFAC). Land use was a major driver on fluvial OM composition at the regional scale of the Meuse Basin, the composition of both fluvial DOM and POM pools showing a shift toward a more microbial/algal and less plant/soil-derived character as human disturbance increased. The comparison of DOM composition between surface and groundwaters demonstrated that this pattern can be attributed in part to the transformation of terrestrial sources by agricultural practices that promote the decomposition of soil organic matter in agricultural lands and subsequent microbial inputs in terrestrial sources. In parallel, human land had contrasting effects on the autochthonous production of DOM and POM. While the in-stream generation of fresh DOM through biological activity was promoted in urban areas, summer autochthonous POM production was not influenced by land use. Finally, soil erosion by agricultural management practices favored the transfer of terrestrial organic matter via the particulate phase. Stable isotope data suggest that the hydrological transfer of terrestrial DOM and POM in human-impacted catchment are not subject to the same controls, and that physical exchange between these two pools of organic matter is limited.