Aiming to characterize the bacterial diversity of modern tufa systems of the Iberian Range (Spain), we surveyed the 16S rRNA gene sequence diversity from 24 sites within three rivers (Añamaza, Mesa and Piedra). These tufas record substantial calcareous growth under different physicochemical conditions and are part of an important, regional landscape-building system. The bacterial community structure and composition, richness and diversity were quantified from denaturing gradient gel electrophoresis fingerprints. Retrieved DNA sequences could be assigned to 10 bacterial phyla and included a variety of phototrophic and heterotrophic groups. Cyanobacteria, mainly filamentous taxa, constituted 43% of all the retrieved sequences, followed by Firmicutes (11%), Gammaproteobacteria (10%), Alphaproteobacteria (7%), Acidobacteria (6%), Bacteroidetes (5%), Betaproteobacteria (4%), Planctomycetes (4%), Actinobacteria (3%) and Deltaproteobacteria (2%). Diatom and Xanthophyceae chloroplast sequences were also detected. Physicochemical variables measured at each site were modelled with multivariate statistics. Principal component analyses yielded the highest variance for salinity-related variables (conductivity; Na+, Cl− and concentrations), which correlated negatively and significantly with diversity indices. However, the highest variance explained by individual principal components was relatively low (< 34%). Overall, we show that these young fluvial tufas are inhabited by a large variety of bacteria in diverse and widespread communities.