The implications of stream flow intermittency for dissolved organic matter (DOM) are not well understood despite its potential significance for water quality and ecosystem integrity. We combined intensive sampling with liquid chromatography and spectroscopic techniques to follow changes in DOC and DON concentrations as well as in DOM size fractions and spectroscopic properties in a temporary stream during an entire contraction–fragmentation–expansion hydrological cycle. DOC and DON concentrations remained low (range = 1.4–5.2 mg C L−1 and 0.05–0.15 mg N L−1) during hydrological contraction and fragmentation, with concomitant increases in the proportion of high molecular weight substances (HMWS) during contraction and of DOM aromaticity during fragmentation. DOC and DON concentrations abruptly increased (up to 8.8 mg C L−1 and 0.37 mg N L−1) at the end of the fragmentation phase, with a concomitant increase in the non-humic, microbial and aquatic character of DOM. Upon rewetting, the DOC and DON concentrations reached their highest values (up to 12.7 mg C L−1 and 0.39 mg N L−1), with concomitant increases in the proportion of HMWS and in the humic, aromatic and terrestrial character of DOM. Subsequently, DOC and DON concentrations recovered to values similar to those at the contraction phase, while DOM composition variables indicated the prevalence of a DOM of humic and terrestrial character during the whole expansion phase. Overall, our results emphasize the importance of hydrological transitions for DOM dynamics in temporary streams, and point to the potential response of perennial streams under future water scarcity scenarios.