ABSTRACTA significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize thein situresponse of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C8to C40) concentrations ranging from 3.1 to 4,500 mg kg−1in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarilyGammaproteobacteria, including representatives of genera with known oil degraders (Alcanivorax,Marinobacter,Pseudomonas, andAcinetobacter). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 107to 10.2 × 107copies g−1) versus clean (0.024 × 107to 1.4 × 107copies g−1) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genusAlcanivoraxshowed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of theGammaproteobacteria(Alcanivorax,Marinobacter) andAlphaproteobacteria(Rhodobacteraceae) as key players in oil degradation there.