The influence of massive crude oil contamination on the microbial population of coastal sediments was investigated in the Cíes Islands 18 and 53 months after the tanker Prestige sank off the NW coast of Spain. Communities were studied by means of culturable and non-culturable methods at three horizons in the sediment (2–5 cm, 12–15 cm and 25–30 cm) in an area heavily affected by the spill. Most probable number of aerobic hydrocarbon degraders was highest in the upper zone and decreased dramatically with depth. Aromatic oxidizing nitrate-reducing bacteria counts were slightly higher than aerobes in the oxidized layer, and also decreased considerably with depth. Iron-reducing bacteria were barely detectable. The highest counts were obtained for sulfate-reducing bacteria, which represented the most relevant fraction of aromatic oxidizers, being maximal at 12–15 cm depth. The community response to high pollution levels was characterized by an increase in culturable populations active towards crude oil components despite the strong decay in the total cell counts. Analysis of whole 16S rRNA gene libraries obtained from the two sampling times and different depths (1460 sequences in all) showed a predominance of Gamma- and Deltaproteobacteria, which was confirmed by fluorescent in situ hybridization. Desulfobacteraceae was the most abundant group among Deltaproteobacteria, followed by sequences affiliated with the order Myxococcales. All retrieved sequences of this order affiliated with a marine myxobacterial clade. Interestingly, sequences affiliated to the order Desulfarculales constituted half of the Deltaproteobacteria sequences retrieved from the heaviest contaminated sample. Principal coordinates analysis of 16S rRNA gene libraries suggested fluctuation in the community distribution with time. Changes in the abundance of certain groups such as Bacteroidetes contributed to these observed differences. Although predominance of certain metabolic types in each horizon could be delimited, a considerable overlap in the use of electron acceptors was observed, confirming that each selected zone could be influenced by more than one respiratory metabolism. Altogether, our results evidence the presence in these sediments of a microbial community with potential to respond against hydrocarbon contamination, consistent with the long pollution history of the site.