5'-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides to their respective nucleosides and phosphate. Most 5'-nucleotidases have broad substrate specificity and are multifunctional enzymes capable of cleaving phosphorus from not only mononucleotide phosphate molecules but also a variety of other phosphorylated metabolites. 5'-Nucleotidases are widely distributed throughout all kingdoms of life and found in different cellular locations. The well-studied vertebrate 5'-nucleotidases play an important role in cellular metabolism. These enzymes are involved in purine and pyrimidine salvage pathways, nucleic acid repair, cell-to-cell communication, signal transduction, control of the ribo- and deoxyribonucleotide pools, etc. Although the first evidence of microbial 5'-nucleotidases was obtained almost 60 years ago, active studies of genetic control and the functions of microbial 5'-nucleotidases started relatively recently. The present review summarizes the current knowledge about microbial 5'-nucleotidases with a focus on their diversity, cellular localizations, molecular structures, mechanisms of catalysis, physiological roles, and activity regulation and approaches to identify new 5'-nucleotidases. The possible applications of these enzymes in biotechnology are also discussed.