Photosynthesis versus irradiance curves and their associated photosynthetic parameters from different phototrophic biofilms isolated from an extreme acidic environment (Río Tinto, SW, Spain) were studied in order to relate them to their species composition and the physicochemical characteristics of their respective sampling locations. The results indicated that the biofilms are low light acclimated showing a photoinhibition model; only floating communities of filamentous algae showed a light saturation model. Thus, all the biofilms analysed showed photoinhibition over 60 µmol photon m−2 s−1 except in the case of Zygnemopsis sp. sample, which showed a light-saturated photosynthesis model under irradiations higher that 200 µmol photon m−2 s−1. The highest values of compensation light intensity (Ic) were showed also by Zygnemosis sp. biofilm (c. 40 µmol photon m−2 s−1), followed by Euglena mutabilis and Chlorella sp. samples (c. 20 µmol photon m−2 s−1). The diatom sample showed the lowest Ic values (c. 5 µmol photon m−2 s−1). As far as we know this is the first attempt to determine the photosynthetic activity of low pH and heavy metal tolerant phototrophic biofilms, which may give light in the understanding of the ecological importance of these biofilms for the maintenance of the primary production of these extreme and unique ecosystems.