Butanol is a promising alternative to fossil-derived fuels. Clostridium genus bacteria are known for their ability to produce butanol as one of the metabolites, however, at the moment this solution is not economically viable. To solve it, the process of butanol production should be optimized. While ABE fermentation has been extensively studied, information about the optimal growth conditions for specific microorganisms often differs from one study to another. Therefore, this study aims to search for optimal growth conditions in sealed serum bottle tests for three widely used strains in ABE fermentation. In this study effects of temperature, pH, and agitation were tested on Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium saccharoperbutylacetonicum. The optimal temperature for C. beijerinckii growth and butanol production was 32 °C, the optimal agitation speed for growth was 0 rpm, but for butanol production, it was 200 rpm. For C. saccharoperbutylacetonicum growth and butanol production pH 7.5, 30 °C temperature and an agitation rate of 100 rpm were optimal, however, this effect was slight. For C. acetobutylicum cultivation optimal temperature, pH, and agitation rate were respectively 37 °C, 6.5, and 200 rpm.