ABSTRACT: The January 2016 Bonnet Carré Spillway (BCS) opening resulted in a large-scale Mississippi River (MR) flood discharge that qualitatively and quantitatively impacted the dissolved organic matter (DOM) cycling in the Lake Pontchartrain Estuary (LPE) located in Louisiana, USA. This early season flood event was a result of the delay of snow formation caused by warmer than normal watershed temperatures. During the diversion period and the subsequent weeks, the LPE water temperature remained lower than pre-flood water temperatures, suppressing carbon cycling. Following that period, the water temperature increased, leading to an increase in the rate of abiotic and biological carbon processing (i.e., mineralization, degradation, and consumption). There were multiple and abnormally high discharges into LPE from the northern tributaries, totaling 43% of the MR flood discharge. As a secondary DOM source, the northern tributaries discharge was qualitatively and quantitatively different from the discharge originating from the river or estuarine sources. The dominant DOM source was determined using satellite images in conjunction with UV-Vis, fluorescence EEMs, and PARAFAC indicators. Overall, the three sources (MR, northern tributaries, and LPE) characteristics were identified by UV-Vis, fluorescence EEMs, and PARAFAC parameters, namely: i) spectral slope (S275), serving as an indicator of lignin-like compounds' molecular weights, with a trend of MR?>?northern tributaries?>?LPE; ii) biological index (BIX), indicating freshness of DOM, with a trend of LPE?>?MR?>?northern tributaries; and iii) Fluorophore T intensity, serving as an indicator of the amount of terrestrial-like sourced DOM, with a trend of northern tributaries?>?LPE?>?MR. It was possible to identify DOM sources and monitor DOM transformation in the water column, increasing our understanding of DOM, carbon, and nitrogen ecological processing.