The Deepwater Horizon oil spill was unprecedented in total loading of petroleum hydrocarbons accidentally released to a marine ecosystem. Controversial application of chemical dispersants presumably accelerated microbial consumption of oil components, especially in warm Gulf of Mexico surface waters. We employed δ¹³C as a tracer of oil-derived carbon to resolve two periods of isotopic carbon depletion in two plankton size classes. Carbon depletion was coincident with the arrival of surface oil slicks in the far northern Gulf, and demonstrated that subsurface oil carbon was incorporated into the plankton food web.