Previous studies have come to contrasting conclusions regarding nutrient limitation of hydrocarbon biodegradation in the Gulf of Mexico, and rate measurements are needed to support oil plume modeling. Thus, this study investigates the rates and controls of biodegradation in seawater and sediments, largely in the deepsea. Sediment and seawater samples were collected on research cruises in the northern Gulf from 2012 to 2014, where the seafloor was impacted by the Deepwater Horizon (DWH) oil spill. Biodegradation was clearly limited by both nitrogen and phosphorus availability in surface waters with significant rates of CO₂ production (100 μmol CO₂ l⁻¹ d⁻¹) only observed in treatments amended with ammonium and phosphate. In deepsea sediments, nutrient amendments resulted in an average of 6 fold higher degradation rates (0.49 μmol CO₂ g sed⁻¹ d⁻¹) compared to unamended controls. Microbial communities responded to oil contamination rapidly in a series of enrichment cultures, and selection was observed for populations of native hydrocarbon-degrading bacteria. Temperature was shown to be a major factor in controlling microbial community composition in the enrichments. At room temperature, community diversity in the enrichments was significantly reduced in the presence of oil, while under 4 °C, the community diversity and evenness remained relatively high upon oil amendment. From the same deepsea sediments, 30 strains of known oil-degrading bacteria (Rhodococcus and Halomonas) were enriched and isolated with hexadecane, phenanthrene, and Macondo oil as the sole carbon and energy source. Detection of these strains in sequence libraries indicates that they may have contributed to the degradation of oil deposited onto the sediments. Rhodococccus strain PC20 degraded approximately one-third of total petroleum hydrocarbons amended into cultures within 7 days. This work elucidates the controls of biodegradation and we provide model pure cultures to further elucidate the ecophysiology of hydrocarbon degradation, focusing on deepsea sediments of the northern Gulf of Mexico.