Microbial and functional diversity were assessed, from a serpentinization-driven subterrestrial alkaline aquifer – Cabeço de Vide Aquifer (CVA) in Portugal. DGGE analyses revealed the presence of a stable microbial community. By 16S rRNA gene libraries and pyrosequencing analyses, a diverse bacterial composition was determined, contrasting with low archaeal diversity. Within Bacteria the majority of the populations were related to organisms or sequences affiliated to class Clostridia, but members of classes Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Deinococci, Gammaproteobacteria and of the phyla Bacteroidetes, Chloroflexi and Nitrospira were also detected. Domain Archaea encompassed mainly sequences affiliated to Euryarchaeota. Only form I RuBisCO – cbbL was detected. Autotrophic carbon fixation via the rTCA, 3-HP and 3-HP/4H-B cycles could not be confirmed. The detected APS reductase alpha subunit – aprA sequences were phylogenetically related to sequences of sulfate-reducing bacteria belonging to Clostridia, and also to sequences of chemolithoautothrophic sulfur-oxidizing bacteria belonging to Betaproteobacteria. Sequences of methyl coenzyme M reductase – mcrA were phylogenetically affiliated to sequences belonging to Anaerobic Methanotroph group 1 (ANME-1). The populations found and the functional key markers detected in CVA suggest that metabolisms related to H2, methane and/or sulfur may be the major driving forces in this environment.