Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.