This study investigates the combined effects of land management and hydrology on the temporal dynamics of dissolved organic matter (DOM) quantity and composition in stream water and groundwaters in an agricultural watershed. We assessed dissolved organic carbon (DOC) concentrations, DOM UV–Vis absorbance, and DOM fluorescence in groundwater under cultivated upland, riparian grassland, and riparian woodland land covers, as well as in the stream water at the watershed outlet and livestock-impacted runoff. During one year, stream water and groundwater were monitored weekly to biweekly, complemented by sub-hourly stream sampling during seven storm events. Results showed that: (1) groundwater DOC concentration was lower in cultivated upland (6.4 ± 5.6 mg l⁻¹) than in riparian grassland and woodland (22.4 ± 13.7 mg l⁻¹ and 17.2 ± 9.9 mg l⁻¹, respectively). (2) The proportion of microbially processed compounds decreased in the order upland cropland > riparian grassland > riparian woodland. (3) Principal component analysis (PCA) of groundwater DOM revealed a change in composition indicating that low-aromaticity microbially processed compounds were preferentially exported to the stream. (4) PCA of stream DOM indicated that seasonal increases in groundwater elevation expanded the contributing source areas, thereby increasing the connectivity between upland croplands and the stream, which amplified the effects of cultivation on fluvial DOM during the winter. (5) Storm events occurring after manure application in spring produced hot moments of manure-derived protein-like DOM transport to streams. Together, these results suggest that cultivated uplands in agricultural lands using animal manure as fertilizer may leach more DOM than vegetative buffers.