During the 2010 Deepwater Horizon oil spill in the Gulf of Mexico, three million liters of chemical dispersants (Corexit 9500 and 9527) were directly applied at the discharging wellhead at 1500m water depth. Such a deep-water large-scale application was unprecedented and the effect of dispersants on oil biodegradation is not yet completely understood. The present study explores the biodegradation of oil, dispersant, dispersed oil or dispersed oil and nutrients at the molecular level using ultra-high resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR–MS) following a laboratory experiment with Gulf deep water. Oil-derived molecular formulae exhibited a specific molecular fingerprint and were mainly observed in the mass range <300Da. The relative abundance of heteroatom-containing (N, S, and P) compounds decreased over time in the oil-only treatments, indicating that they may have served as nutrients when oil-derived hydrocarbons were metabolized. Relative changes over time in the molecular composition were less pronounced in the dispersed oil treatments compared to the oil-only treatments, suggesting that dispersants affected the metabolic pathways of organic matter biodegradation. In particular, dispersant addition led to an increase of S-containing organic molecular formulae, likely derived from the surfactant di-octyl sulfosuccinate (DOSS). DOSS and several dispersant-derived metabolites (with and without S) were still detectable after six weeks of incubation, underscoring that they were not rapidly biodegraded under the experimental conditions. FT-ICR–MS fragmentation studies allowed tentatively assigning structures to several of these molecules, and we propose that they are degradation products of DOSS and other dispersant components. The present study suggests preferential degradation, transformation and enrichment of distinct dispersant molecules, highlighting the need to include these compounds when tracking Corexit-derived compounds in the environment.