Carbonate microbialites in lakes can serve as valuable indicators of past environments, so long as the biogenicity and depositional setting of the microbialite can be accurately determined. Late Pleistocene to Early Holocene frondose draping tufa deposits from Winnemucca Dry Lake (Nevada, USA), a subbasin of pluvial Lake Lahontan, were examined in outcrop, petrographically, and geochemically to determine whether microbially induced precipitation is a dominant control on deposition. These observations were compared to modern, actively accumulating microbialites from Fayetteville Green Lake (New York, USA) using similar methods. In addition, preserved microbial DNA was extracted from the Lahontan tufa and sequenced to provide a more complete picture of the microbial communities. Tufas are texturally and geochemically similar to modern thrombolitic microbialites from Fayetteville Green Lake, and the stable isotopic composition of organic C, N, inorganic C, and O supports deposition associated with a lacustrine microbial mat environment dominated by photosynthetic processes. DNA extraction and sequencing indicate that photosynthetic microbial builders were present during tufa deposition, primarily Chloroflexi and Proteobacteria with minor abundances of Cyanobacteria and Acidobacteria. Based on the sequencing results, the depositional environment of the tufas can be constrained to the photic zone of the lake, contrasting with some previous interpretations that put tufa formation in deeper waters. Additionally, the presence of a number of mesothermophilic phyla, including Deinococcus–Thermus, indicates that thermal groundwater may have played a role in tufa deposition at sites not previously associated with groundwater influx. The interpretation of frondose tufas as microbially influenced deposits provides new context to interpretations of lake level and past environments in the Lahontan lake basins.