The global demand for crude oil is expected to continue to rise in future while simultaneously oil production is currently reaching its peak. Subsequently, rising oil prices and their negative impacts on economy, together with an increased environmental awareness of our society, directed the focus also on the biotechnological production of fuels. Although a wide variety of such fuels has been suggested, only the production of ethanol and biodiesel has reached a certain economic feasibility and volume, yet. This review focuses on the current state and perspectives of biotechnological production of biodiesel-like compounds. At present by far most of the produced biodiesel is obtained by chemical transesterification reactions, which cannot meet the demands of a totally ‘green’ fuel production. Therefore, also several biotechnological biodiesel production processes are currently being developed. Biotechnological production can be achieved by purified enzymes in the soluble state, which requires cost-intensive protein preparation. Alternatively, enzymes could be immobilized on an appropriate matrix, enabling a reuse of the enzyme, although the formation of by-products may provide difficulties to maintain the enzyme activity. Processes in presence of organic solvents like t-butanol have been developed, which enhance by-product solubility and therefore prevent loss of enzyme activity. As another approach the application of whole-cell catalysis for the production of fatty acid ethyl esters, which is also referred to as ‘microdiesel’, by recombinant microorganisms has recently been suggested.