We studied the biological activity and prokaryotic diversity associated with 2 samples of different physico-chemical characteristics in a pristine coastal aquifer at Doñana National Park, south-western Spain. Sulphate reduction, denitrification and iron-metabolising activities were detected in the aquifer, as well as different enzymatic activities related to the degradation of labile and complex pools of organic matter. Prokaryotic diversity was assessed by environmental 16S rRNA gene amplification, cloning and sequencing. Bacterial diversity was greater in the shallower aquifer sample (49S1, 15 m depth), including members of the Proteobacteria, Acidobacteria, Planctomycetes, Nitrospirae, some divergent lineages and the candidate divisions SPAM, OP3, OP11, 'Endomicrobia' or Termite Group 1 and a novel division-level group including denitrifying bacteria associated to anaerobic methane-oxidising archaea, whereas only Proteobacteria and Firmicutes were detected in the deeper sample (56S, 80 m depth). By contrast, archaea seemed much more diverse in the deeper aquifer sample, with members of the Methanomicrobiales, ANME2-related Methanosarcinales and other divergent lineages, whereas only Group I Crenarchaeota were detected in the shallower sample. Betaproteobacteria were the most abundant and diverse group in both sample libraries, together with the Gammaproteobacteria in the deeper and more saline 56S sample. We detected microorganisms potentially involved in carbon, sulphur and nitrogen cycling. Interestingly, members of both aerobic (Alpha-and Gammaproteobacteria) and anaerobic methane oxidisers (ANME2 archaea) were found in the same aquifer sample.