Lake Clifton in Western Australia is recognized as a critically endangered ecosystem and the only thrombolite reef in the southern hemisphere. There have been concerns that increases in salinity and nutrient run-off have significantly impacted upon the thrombolite microbial community. Here we used cultivation-independent molecular approaches to characterize the microbial diversity of the thrombolites at Lake Clifton. The most dominant phyla currently represented are the Proteobacteria with significant populations of Bacteroidetes and Firmicutes. Cyanobacteria, previously invoked as the main drivers of thrombolite growth, represent only a small fraction (~1–3% relative abundance) of the microbial community. We report an increase in salinity and nitrogen levels at Lake Clifton that may be contributing to a change in dominant microbial populations. This heightens concerns about the long-term health of the Lake Clifton thrombolites; future work is needed to determine if phyla now dominating this system are capable of the required mineral precipitation for continued thrombolite growth.