In the aftermath of the Deepwater Horizon oil spill in 2010, there was concern about the risk to human health through consumption of contaminated seafood from the region. The National Oceanic and Atmospheric Administration (NOAA), in collaboration with U.S. Food and Drug Administration (FDA), the Environmental Protection Agency, and Gulf Coast States, worked together to ensure that seafood from the Gulf of Mexico was safe to eat by developing seafood safety criteria, monitoring procedures, re-opening protocols, as well as seafood surveillance monitoring plans. As part of this seafood safety assessment, edible tissues of seafood collected in state and federal waters were tested for polycyclic aromatic hydrocarbons (PAHs) and dispersants using sensory testing, as well as chemical analyses. To increase the analytical capacities of laboratories testing seafood from the Gulf, the FDA developed and validated high-performance liquid chromatography/fluorescence and liquid chromatography-mass spectrometry methods (in conjunction with NOAA) that accurately and precisely measured chemical compounds, including dispersants, associated with this oil spill event. Thousands of seafood samples collected during reopening and surveillance in the Gulf, as well as those obtained dockside and in the marketplace, have been analyzed using these methods. This dataset includes information on levels of polycyclic aromatic hydrocarbons and dispersant component dioctyl sodium sulfosuccinate in edible tissues of commercially and recreationally important seafood species collected in Federal waters in the aftermath of the Deepwater Horizon Oil Spill.