Microorganisms inhabit almost every imaginable environment and are important players in biogeochemical cycles. With the development and improvement of molecular biology-based approaches, the functions and community composition of microorganisms in ecosystems can be explored in more detail than ever before. Rapidly advancing molecular techniques have been applied to questions regarding microbial diversity, biogeography, and responses to environmental changes. Studies of microorganisms in the environment generally focus on three objectives-determining which microorganisms are present, what their functions are, and which are active at a given time. Comprehending the range of techniques currently available can be daunting. To facilitate the selection of the appropriate approach by researchers to study microbial communities in the environment, we introduce molecular methods according to microbial diversity and function, such as the rapidly developing high-throughput sequencing, meta-omics, and microbial single-cell approaches (e.g., nano-scale secondary ion mass spectrometry-fluorescence in situ hybridization, NanoSIMS-FISH), and their applications in microbial ecology studies.