Modern microbial mats rarely lithify or preserve their layering except under exceptional circumstances of elevated calcium carbonate (CaCO3) concentrations or reduced grazing/bioturbation pressures. Stromatolite (layered accretions formed by microbial activity) lithification and CaCO3 precipitation are dependent on the interplay between autotrophic and heterotrophic microbial mat communities. Key processes, such as nutrient uptake and primary production by the autotrophic community (cyanobacteria and microalgae) are hypothesised to influence the precipitation process and overall stromatolite growth. As this has yet to be demonstrated, this study investigated primary production, nutrient uptake dynamics (N and P) and CaCO3 precipitation of tufa stromatolites by conducting in situ experiments in a well-studied system near Port Elizabeth, South Africa. There was limited variability between stromatolite pool locations in terms of inorganic content, calcification rates and primary production. Importantly, this demonstrates that these bottom-up processes are likely not solely responsible for observed gradients of stromatolite biomass, as previously thought. This suggests that stromatolite microalgae, when exposed to suitable conditions (i.e. sufficient nutrients and carbonate saturation), are adapted towards an optimum level of accretion to maximise competitive utilisation of these resources. This has important implications for interpreting both modern and past stromatolite growth conditions. Differential stromatolite growth conditions/biomass is likely instead a function of the different durations under which saline, brackish or freshwater (which affect nutrient and carbonate dynamics) are experienced between pool locations.