Abstract Date palm empty fruit bunch (EFB) is a reliable and underutilized agriculture residue generated by the date industry. EFB contains polymeric sugars that can be converted to biofuel when subjected to pretreatment, enzymatic hydrolysis, and microbial fermentation. Optimized biofuel processing conditions produced 119 kg ethanol per dry metric ton EFB. The highest sugar conversions from enzymatic hydrolysis were found with Ammonia Fiber Expansion (AFEX™, MBI International, Lansing, MI) pretreatment conditions of 2:1 ammonia-to-biomass ratio, 80% moisture content, and a temperature of 120°C, for 30 min residence time. These conditions produced 52.5 g/L and 34.8 g/L of fermentable glucose and xylose, respectively. This hydrolysate was inoculated with a glucose- and xylose-consuming recombinant yeast strain producing 35.8 g/L and 31.8 g/L ethanol with and without nutrient supplementation, respectively. A complete mass balance for EFB pretreatment, enzymatic hydrolysis, and microbial fermentation is pres...