Nowadays, with increasing hunger for liquid fuels usable in transportation, alternatives to crude oil derived fuels are being searched very intensively. In addition to bioethanol and ethyl or methyl esters of rapeseed oil that are currently used as bio-components of transportation fuels in Europe, other options are investigated and one of them is biobutanol, which can be, if produced from waste biomass or non-food agricultural products, classified as the biofuel of the second generation. Although its biotechnological production is far more complicated than bioethanol production, its advantages over bioethanol from fuel preparation point of view i.e. higher energy content, lower miscibility with water, lower vapour pressure and lower corrosivity together with an ability of the producer, Clostridium bacteria, to ferment almost all available substrates might outweigh the balance in its favour. The main intention of this chapter is to summarize briefly industrial biobutanol production history, to introduce the problematic of butanol formation by clostridia including short description of various options of fermentation arrangement and most of all to provide with complex fermentation data using little known butanol producers Clostridium pasteurianum NRRL B-592 and Clostridium beijerinckii CCM 6182. A short overview follows concerning the use of biobutanol as a fuel for internal combustion engines with regard to properties of biobutanol and its mixtures with petroleum derived fuels as well as their emission characteristics, which are illustrated based on emission measurement results obtained for three types of passenger cars.