Calcification of freshwater streams and the involvement of microorganisms in this process are still not fully understood. Here, we report on the metagenomic and metatranscriptomic analyses of bacterial community structures derived from an actively calcifying karst water creek biofilm and underlying tufa by employing next-generation sequencing technologies.The persistent and metabolically active bacterial communities were assessed by DNA-based and RNA-based 16S rRNA gene sequence analysis, respectively. We identified filamentous Cyanobacteria belonging to the Oscillatoriales as the predominant bacterial microorganisms in the biofilm. Cyanobacteria were accompanied by a high diversity of mainly aerobic members affiliated to different bacterial phyla. The second most abundant phylum was Proteobacteria, represented by the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria. In addition, Chloroflexi, Planctomycetes, Actinobacteria, Acidobacteria, Bacteroidetes, Spirochaetes, and Verrucomicrobia were present in higher abundances (>0.5%). Several of these phyla included potentially novel subgroups. The tufa stromatolite carbonate one centimeter below the surface exhibited a similar prokaryotic community as the superficial biofilm but showed a lower abundance of Cyanobacteria and a more diverse microbial community. We obtained insights into the in situ microbial metabolism by employing directed sequencing of enriched mRNA (cDNA) and subsequent taxonomic and functional analysis of biofilm-derived reads. High activities in photosynthesis, carbon and protein metabolism were indicated.