The microbial diversity of the water column and the sediments of an athalassohaline environment, Tirez Lagoon (La Mancha, Spain) have been characterized using culture-dependent and – independent methods. Algae (Diatoms and Dunaliella) and Cyanobacteria (Microcoleus, Nodularia, Anabaena, Nostoc and Pseudobaena) were the main photosynthetic primary producers detected in the ecosystem. A Polarella-like dinoflagelate was detected in high numbers in the water column. In addition to the expected Haloarchaea (Halobacterium, Haloarcula, Halorubrum, Halomicrobium, Natronobacterium and Natronomonas genera) the highest number of phylotypes corresponded to members of the bacterial Phylum Bacteroidetes (Psychroflexus sp., Salegenibacter sp., Flavobacterium sp., Marinilabilia sp., Muricauda sp. and Microscilla sp.) as has been described for other athalassohaline environments. Of the Proteobacteria Phylum the highest diversity corresponded to the Gammaproteobacteria (Halomonas sp., Marinobacter sp., Idiomarina sp., Halochromatium sp., Methylophaga sp. and Thiomicrospira sp.) and Deltaproteobacteria (Desulfohalobium sp., Desulfonema sp., Desulfobacterium sp., Desulfonatronovibrio sp. and Syntrophobacter sp.). Also members of the Betaproteobacteria (Thiobacillus sp.) and Alphaproteobacteria (Rhodobacter sp.) were detected in the sediments. Gram positive microorganisms of the phyla Firmicutes (Bacillus sp. and Desulfotomaculum sp.) and Actinobacteria (Arthrobacter sp.), also detected in other athalassohaline environments, have been isolated and identified from Tirez samples. In addition to the Haloarchaea, methanogens (Methanolobus sp., Methanohalobium sp. and Methanoculleus sp.) and sulfate reducing Archaea (Archaeoglobus sp.) have been detected in the anoxic and reductive sediments of Tirez lagoon. The comparison with other athalassohaline environments allowed the cosmopolitan/ubiquitous (not properly used) activities (Haloarchaea, Bacteroidetes, Deltaproteobacteria, Firmicutes and Actinobacteria) to be identified. Although sulfate reducing bacteria and methanogens have been described previously in thalassohaline environments, this is the first time that the microorganisms responsible for these activities have been identified in an athalassohaline ecosystem.