Microbialites with laminar (stromatolite) and thrombolitic (thrombolite) fabrics are ubiquitous within the Cenozoic freshwater sedimentary record. However, the biology and physiology of the living prokaryote–microphyte biofilms which produced them is only now becoming understood. The present contribution describes a flowing water experimental mesocosm study spanning over 2·5 years and run under near-natural conditions. This work focussed on microbial biofilm precipitation mechanisms which produce thrombolitic carbonate micro-fabrics capable of preservation in the geological record. In particular, the roles of microbial guilds and carbonate precipitation processes were examined and recorded at all stages of thrombolite development. The mesocosm experiments convincingly demonstrated that the biofilm community actively encouraged calcium ion precipitation derived from flowing waters. This precipitation took the form of amorphous calcium carbonate nanosphere clusters. These clusters were not randomly distributed within the biofilm extracellular polymeric substances but were focussed in the close vicinity of living filament and coccoid bacterial clusters within individual living biofilm layers. Significantly, the precipitates never replaced microbial cell walls and never buried the living microbes. During nanosphere precipitation extracellular polymeric substances were progressively occluded from between the developing nanosphere clusters. However, extracellular polymeric substances were never totally removed from within the amorphous calcium carbonate clusters until they had neomorphosed into microspar crystals. The orientation of precipitating microspar crystals within the biofilm appeared to be controlled by the host extracellular polymeric substance fabric (cf. typical crystal growth from solid substrates). Precipitates were organized around the margins of a cancellate microfabric developed by a range of microbial guilds within each biofilm layer. This produced a distinct thrombolitic fabric within the biofilm which was quite distinct from laminar stromatolite fabrics. It is concluded that the mesocosm grown freshwater biofilms and their associated microbialite calcite micro-fabrics present a universally applicable model. Importantly, they provide a mechanism for thrombolite micro-fabric developments throughout the geological record.