Reports of cyanobacterial blooms in temperate lakes have been increasing over the last few decades. If blooms are indeed becoming more frequent or intense, this poses a problem for water and ecosystem management as blooms can be toxic to wildlife and humans. Here we used a paleolimnological approach to determine whether cyanobacteria have been increasing in Western Quebec, a region with thousands of lakes and a lack of historical surface water monitoring data. We compared lakes within and outside of Gatineau Park, a protected conservation area since 1938. Sediment cores dating back to pre-European settlement of the region were analyzed for temporal trends in cyanobacteria, in order to assess the effect of land use change and/or climate change before and after the Park’s creation. We extracted sediment DNA and analyzed for the 16S rRNA gene specific to cyanobacteria based on a qPCR assay for absolute gene copy numbers. These results were compared with analyses of the carotenoid pigments zeaxanthin and echinenone, specific to cyanobacteria, along with analyses of diatoxanthin and β-carotene, representative of diatoms and all algae respectively. Overall, gene copy numbers of cyanobacterial 16S rRNA pointed to a significant increase in cyanobacteria in all five lakes over the past 30 years and also since the Park’s creation, when compared to the historical average (past 150 years). In contrast, qPCR analyses of eubacterial gene copies for glutamine synthesis indicated that total microbial abundance exhibited a relatively smaller change over the same time periods. No significant difference in the percent increase of cyanobacteria, quantified by both gene copies and carotenoid pigments, was observed between lakes within and outside of the Park. This would suggest that other factors, such as a warming climate documented in this region, may be driving the increase in cyanobacteria. Sediment DNA has the potential to corroborate more classical fossil remains and provide novel information on microbial structure and function of past ecosystems.