Dispersant application is a primary emergency oil spill response strategy and yet the efficacy and unintended consequences of this approach in marine ecosystems remain controversial. To address these uncertainties, ex situ incubations were conducted to quantify the impact of dispersant on petroleum hydrocarbon (PHC) biodegradation rates and microbial community structure at as close as realistically possible to approximated in situ conditions [2 ppm v/v oil with or without dispersant, at a dispersant to oil ratio (DOR) of 1:15] in surface seawater. Biodegradation rates were not substantially affected by dispersant application at low mixing conditions, while under completely dispersed conditions, biodegradation was substantially enhanced, decreasing the overall half-life of total PHC compounds from 15.4 to 8.8 days. While microbial respiration and growth were not substantially altered by dispersant treatment, RNA analysis revealed that dispersant application resulted in pronounced changes to the composition of metabolically active microbial communities, and the abundance of nitrogen-fixing prokaryotes, as determined by qPCR of nitrogenase (nifH) genes, showed a large increase. While the Gammaproteobacteria were enriched in all treatments, the Betaproteobacteria and different families of Alphaproteobacteria predominated in the oil and dispersant treatment, respectively. Results show that mixing conditions regulate the efficacy of dispersant application in an oil slick, and the quantitative increase in the nitrogen-fixing microbial community indicates a selection pressure for nitrogen fixation in response to a readily biodegradable, nitrogen-poor substrate.