Fluorescent in situ hybridization (FISH) on environmental samples has become a standard technique to identify and enumerate microbial populations. However, visualization and quantification of cells in environmental samples with complex matrices is often challenging to impossible, and downstream protocols might also require the absence of organic and inorganic particles for analysis. Therefore, quite often microbial cells have to be detached and extracted from the sample matrix prior to use in FISH. Here, details are given for a routine protocol to extract intact microbial cells from environmental samples using density gradient centrifugation. This protocol is suitable and adaptable for a wide range of environmental samples.