The dissolved inorganic carbon (DIC) acquisition mechanisms were researched in intact microbial mats dominated by the cyanobacteria Microcoleus chthonoplastes Thuret, by determining the effect on photosynthesis of different inhibitors. The microbial mats exhibited high affinity for DIC at alkaline pH, with Km(DIC) values similar to the ones described for pure cultures of cyanobacteria and algae in which carbon concentrating mechanisms have been researched. Besides, the photosynthesis was non‐sensitive to pH changes within the range of 5.6–9.6, indicating that HCO3− was the main DIC source used for photosynthesis. The M. chthonoplastes mats featured external and internal carbonic anhydrase (CA) activity as measured in intact cells and cell extracts, respectively. Acetazolamide (AZ, which slowly enters the cell and then inhibits mainly the external CA) and ethoxyzolamide (EZ, which inhibits both external and internal CA) reduced significantly the oxygen evolution rates, demonstrating that the CA was implied in the DIC acquisition. Vanadate inhibited photosynthesis by 60% although its application, when CA being inhibited (i.e. after applying AZ + EZ), did not produce any additional effect. It could indicate that ATPase‐dependent HCO3− use occurred and also that this putative mechanism was coupled with CA‐like activity at the plasma membrane. The involvement of Na+‐dependent HCO3− transporters in DIC acquisition was also inferred as monensin and 4‐4′‐diisothiocyanatostibilene‐2,2′‐disulfonate (DIDS) reduced photosynthesis by 70%. DIDS produced a strong inhibitory effect even after application of AZ + EZ + vanadate, indicating that this mechanism was not related to CA activity. The microbial mats become subject to very unfavourable conditions for Rubisco carboxylation at their natural habitats (e.g. external pH of 10.5 and O2 concentration doubled with respect to saturation concentration); therefore, this putative diversity of DIC acquisition mechanisms could ensure their growth under these extreme conditions.