Burgeoning scientific literature proves that enzymes can catalyze chemical reactions in non-conventional media, are active against liquid, solid or gaseous substrates, retain their catalytic function in organic solvents, biphasic systems composed of organic solvent and water and supercritical fluids. Non-aqueous media appeared to be a promising alternative as an environment for the reactions catalyzed by enzymes. The possibility of carrying out enzymatic processes in such unusual milieus enlarged the range of applications of biocatalysis, and solved many problems witnessed by pharmaceutical, food and chemical industries. The paper describes both advantages and disadvantages of non-conventional media, and presents the examples of practical uses of selected enzymes (lipases, proteases, oxidoreductases, glycosidases) in these systems. The details on medium composition and the form of the biocatalyst are included.