This study determined the natural abundance isotopic compositions (13C, 14C) of the primary carbon pools and microbial communities associated with modern freshwater microbialites located in Pavilion Lake, British Columbia, Canada. The Δ14C of dissolved inorganic carbon (DIC) was constant throughout the water column and consistent with a primarily atmospheric source. Observed depletions in DIC 14C values compared with atmospheric CO2 indicated effects due either to DIC residence time and/or inputs of 14C-depleted groundwater. Mass balance comparisons of local and regional groundwater indicate that groundwater DIC could contribute a maximum of 9–13% of the DIC. 14C analysis of microbial phospholipid fatty acids from microbialite communities had Δ14C values comparable with lake water DIC, demonstrating that lake water DIC was their primary carbon source. Microbialite carbonate was also primarily derived from DIC. However, some depletion in microbialite carbonate 14C relative to lake water DIC occurred, due either to residence time or mixing with a 14C-depleted carbon source. A detrital branch covered with microbialite growth was used to estimate a microbialite growth rate of 0.05 mm year−1 for the past 1000 years, faster than previous estimates for this system. These results demonstrate that the microbialites are actively growing and that the primary carbon source for both microbial communities and recent carbonate is DIC originating from the atmosphere. While these data cannot conclusively differentiate between abiotic and biotic formation mechanisms, the evidence for minor inputs of groundwater-derived DIC is consistent with the previously hypothesized biological origin of the Pavilion Lake microbialites.