Abstract A mixed culture was constructed from compost of Napiergrass and sheep dung under anaerobic thermophilic conditions (60 °C). The native microflora was cultivated for numerous generations to obtain a stable mixed culture that can degrade lignocelluloses. The fifth generation of the mixed culture consisting of five main bacteria (Clostridium strain TCW1, Bacillus sp. THLA0409, Klebsiella pneumoniae THLB0409, Klebsiella oxytoca THLC0409, and Brevibacillus strain AHPC8120) was employed to investigate the effects of operating conditions on culture growth and production of biochemical products, including ethanol. The mixed culture effectively degraded a diverse range of lignocellulosic materials, including microcrystalline cellulose (avicel) and natural lignocelluloses (Napiergrass). Acetic acid, ethanol, and butanol were the main biochemical products produced by biological fermentation. Under optimal conditions, ethanol yields from avicel and Napiergrass reached maxima of 0.108 and 0.040 g g−1, representing ethanol productivities of 0.00055 and 0.00028 g g−1 h−1, respectively.