Microbial cholesterol oxidase is an enzyme of great commercial value, widely employed by laboratories routinely devoted to the determination of cholesterol concentrations in serum, other clinical samples, and food. In addition, the enzyme has potential applications as a biocatalyst which can be used as an insecticide and for the bioconversion of a number of sterols and non-steroidal alcohols. The enzyme has several biological roles, which are implicated in the cholesterol metabolism, the bacterial pathogenesis, and the biosynthesis of macrolide antifungal antibiotics. Cholesterol oxidase has been reported from a variety of microorganisms, mostly from actinomycetes. We recently reported cholesterol oxidases from gram-negative bacteria such as Burkholderia and Chromobacterium. These enzymes possess thermal, detergent, and organic solvent tolerance. There are two forms of cholesterol oxidase, one containing a flavin adenine dinucleotide cofactor non-covalently bound to the enzyme (class I) and the other containing the cofactor covalently linked to the enzyme (class II). These two enzymes have no significant sequence homology. The phylogenetic tree analyses show that both class I and class II enzymes can be further divided into at least two groups.