Recent studies have shown that even in the same population, there might be significant differences among individual cells at both cellular and molecular levels, indicating multiple levels of heterogeneity in microbial cells. Meanwhile, depending on establishing pure culture in the laboratory for the targeted microbes, traditional microbiology approaches are unable to present the original state of microorganisms in natural environments. Moreover, so far only small number of microbial species is cultivated successfully in the laboratory, leaving a great deal of microbial information untouched. Single-cell microbiology can be effective tools for addressing these issues and providing better and in-depth understanding of state of microbial cells. In this mini-review, we briefly introduce the significance of single-cell microbiology, and summarize current state-of-the-art methodologies in this new research area, especially the emerging single-cell omics tools for microbial research.