Due to usage of fossil fuels, the depletion of world crude oil reserves and increased deteriorating climate conditions have reached a high level. These circumstances have led researches to search for alternative and efficient fuels. The main biofuels considered are bioethanol and biodiesel. In this study, ethanol and biodiesel production from cotton stalk and seed were aimed using liquid hot water (LHW) along with consecutive processes, where separate saccharification and fermentation (SHF) process was carried out. The maximum ethanol concentrations of 0.348 g/L and 0.721 g/L were achieved at 24 h and 72 h, respectively. For biodiesel conversion, cottonseed oil was subjected to transesterification, where the main interest was to utilize the by-product, glycerol. Three different glycerol concentrations were investigated in terms of ethanol fermentation using Escherichia coli K1 active culture. The maximum ethanol concentration of 0.415 g/L was achieved at 20 mL glycerol concentration for 48 h. Overall, cotton stalk and seed have the potential to be utilized on an industrial scale.