Microorganisms are ubiquitous and represent a taxonomically and functionally diverse component of freshwater environments of significant ecological importance. The bacteria, archaea, and microbial eukarya in freshwater systems support a range of ecosystem processes and functions, including mediating all major biogeochemical cycles, and therefore regulate the flow of multiple ecosystem services. Yet relative to conspicuous higher taxa, microbial ecology remains poorly understood. As the anthropocene progresses, the demand for freshwater–ecosystem services is both increasing with growing human population density, and by association, increasingly threatened from multiple and often interacting stressors, such as climate change, eutrophication, and chemical pollution. Thus, it is imperative to understand the ecology of microorganisms and their functional role in freshwater ecosystems if we are to manage the future of these environments effectively. To do this, researchers have developed a vast array of molecular tools that can illuminate the diversity, composition, and activity of microbial communities. Within this primer, we discuss the history of molecular approaches in microbial ecology, and highlight the scope of questions that these methods enable researchers to address. Using some recent case studies, we describe some exemplar research into the microbial ecology of freshwater systems, and emphasize how molecular methods can provide novel ecological insights. Finally, we detail some promising developments within this research field, and how these might shape the future research landscape of freshwater microbial ecology.