Rice straw (RS) is one of the lignocellulosic wastes with the highest global production. The main objective of this study was to maximise the butanol production by Clostridium beijerinckii DSM 6422 from RS pretreated by microwave-assisted hydrothermolysis. Two different fermentation strategies were compared: separate hydrolysis and fermentation (SHF, two-step process) and simultaneous saccharification and fermentation (SSF, one-step process). In parallel, the variables that significantly affected the butanol production were screened by using fractional factorial designs. Butanol concentration and productivity at 48 h were, respectively, 8% and 173% higher in SSF than in SHF. A one-step process was more efficient than a two-step process, especially considering the time savings derived from much higher productivity. From these results, SSF was further optimised by response surface methodology with central composite design over the key factors on the butanol production at 48 h: initial pH, enzyme loading and yeast extract concentration. The optimum point yielded a butanol productivity of 0.114 g L-1h−1, with a butanol-biomass ratio of 51 g kg−1 of raw RS (ABE-biomass ratio of 77.0 g kg−1 of raw RS). The parameter with the greatest effect was enzyme loading, with an optimal value of 13.5 FPU g-dw-1. This study showed that microwave-processed RS has great potential as a substrate for the butanol production from ABE fermentation when combining process stages by SSF.