The ice-covered lakes in the McMurdo Dry Valleys (MDV) of Antarctica provide end-member ecosystems for understanding the production of dissolved organic matter (DOM) in aquatic ecosystems in the absence of vegetation on the landscape and under resource and nutrient constraints. Given these constraints, DOM in MDV lakes is derived solely from microbial phototrophs and heterotrophic bacteria, contrasting with the dominant terrestrial sources in temperate regions. Previous research developed fluorometric approaches for characterizing DOM, including in MDV lakes. In this study we leveraged these approaches along with contemporary molecular-based techniques to elucidate changes in DOM composition across the depth profile for Lake Fryxell in the MDV. The results showed that the presence of organic molecules containing sulfur increased at depth where anoxic conditions prevailed. To evaluate the influences of climate-induced rising lake levels and multiple flood events in the MDV, we compared recent and historical samples. The results indicated a remarkable consistency in source-related fluorescence metrics over time, whereas a twofold decrease in sulfur content of the fulvic acid fraction was observed in samples from above and below the oxycline. Biogeochemical processes associated with the influx of iron oxide–rich sediments during flood events may have contributed to this change, and similar processes may stimulate biogeochemical cycling and remineralization in temperate lakes during seasonal transitions.