The influence of extracellular polymeric substances on carbonate mineral growth in natural settings remains one of the most poorly understood contributors to the growth of non-marine carbonate sediments. The influences of these materials are complicated by their association with living cells creating local microenvironments via metabolism and enzyme production, and by our uncertainty about the extracellular polymeric substances materials themselves. Different mixtures of extracellular polymeric substance molecules may behave in different ways, and differences in the local physical environment may alter how the mixtures influence mineral formation, and even result in different patterns of polymerisation. Here, the influence of extracellular polymeric substances on calcite precipitation rate and Mg/Cacalcite in the absence of cells is investigated using extracts of extracellular polymeric substances from temperate fluvial tufa biofilm. The influence is complex, with the concentration of extracellular polymeric substances in solution altering deposition rate and trace element incorporation. Moreover, the results show interaction of EPS presence/ absence and both temperature and salinity. However, despite extracting extracellular polymeric substances from the same parent sample, a uniform influence was not found in these experiments, implying that the mixture is sufficiently variable within a sample for microenvironments within the biofilm to either promote or inhibit mineralisation. As sedimentologists, we can no longer take the view that extracellular polymeric substances is a bystander material, or that it has a single set of coherent and predictable or intuitive influences. Rather, the emphasis must be on investigating the specific mixtures present in nature, and their complex and dynamic interaction with both mineral surfaces and hydrochemical conditions.