During theDeepwater Horizonoil well blowout in the Gulf of Mexico, the application of 7 million liters of chemical dispersants aimed to stimulate microbial crude oil degradation by increasing the bioavailability of oil compounds. However, the effects of dispersants on oil biodegradation rates are debated. In laboratory experiments, we simulated environmental conditions comparable to the hydrocarbon-rich, 1,100 m deep plume that formed during theDeepwater Horizondischarge. The presence of dispersant significantly altered the microbial community composition through selection for potential dispersant-degradingColwellia,which also bloomed in situ in Gulf deep waters during the discharge. In contrast, oil addition to deepwater samples in the absence of dispersant stimulated growth of natural hydrocarbon-degradingMarinobacter.In these deepwater microcosm experiments, dispersants did not enhance heterotrophic microbial activity or hydrocarbon oxidation rates. An experiment with surface seawater from an anthropogenically derived oil slick corroborated the deepwater microcosm results as inhibition of hydrocarbon turnover was observed in the presence of dispersants, suggesting that the microcosm findings are broadly applicable across marine habitats. Extrapolating this comprehensive dataset to real world scenarios questions whether dispersants stimulate microbial oil degradation in deep ocean waters and instead highlights that dispersants can exert a negative effect on microbial hydrocarbon degradation rates.