Microbes thrive and, in turn, influence the earth's environment, but most are poorly understood because of our limited capacity to reveal their natural diversity and function. Developing novel tools and effective strategies are critical to ease this dilemma and will help to understand their roles in ecology and human health. Recently, droplet microfluidics is emerging as a promising technology for microbial studies with value in microbial cultivating, screening, and sequencing. This review aims to provide an overview of droplet microfluidics techniques for microbial research. First, some critical points or steps in the microfluidic system are introduced, such as droplet stabilization, manipulation, and detection. We then highlight the recent progress of droplet-based methods for microbiological applications, from high-throughput single-cell cultivation, screening to the targeted or whole-genome sequencing of single cells.