A new enzymatic technique for the detachment of bacteria from soil particles was developed and applied to different soil samples taken at various sampling sites and depths. Many soil microorganisms are closely associated with the organic matrix of soil particles. They produce extracellular polymeric substances (EPS), which promote the irreversible adhesion of cells to soil particulates. To characterize the EPS, a prestaining of the soil samples with different lectins was performed. Samples from a sewage field, an urban park, a farmland, a mixed forest and garden mold were stained with a set of FITC-labelled lectins from Triticum vulgaris, Ulex europaeus, Concanavalin A and Pseudomonas aeruginosa. Based on the results, a combination of alpha-glucosidase, beta-galactosidase and a lipase was chosen for degradation of the EPS structures, followed by gentle mechanical and chemical dispersion in a modified sodium pyrophosphate buffer. The samples were fixed with formaldehyde and total cell counts were determined by DAPI staining. With the exception of the wheat field sample, this technique revealed up to 22-fold higher total cell counts for all investigated soil samples compared to the conventional detachment method, a simple dispersion with sodium pyrophosphate buffer. Efficiency of the technique was assessed by scanning electron microscopy. These images showed convincingly that the enzymatic treatment followed by sonication efficiently detached the bacteria and left the soil particles almost blank.