The current study encompasses the development of a combined pretreatment strategy using deep eutectic solvent (DES) i.e. choline chloride (ChCl)/glycerol and ultrasonic waves for the digestibility of sugarcane bagasse (SCB). Pretreated biomass was saccharified by an enzyme cocktail produced in house from a novel fungus Aspergillus assiutensis VS34. Combined ChCl/glycerol and ultrasound pretreatment yielded higher sugars (276.8 mg/g biomass) than either of the individual pretreatments i.e. ChCl/glycerol (235.3 mg/g biomass) or ultrasound (174.5 mg/g biomass). Optimization of pretreatment process variables (biomass loading, sonication amplitude and pretreatment time) by Design of Experiment enhanced sugar yield by 1.12-fold. Sugar hydrolysate obtained was fermented by dual yeast cultures i.e. Saccharomyces cerevisiae and Pichia stipitis to get an ethanol yield of 142.09 mg/g biomass. To understand the molecular mechanism of combined DES/ultrasound pretreatment which led to eased hydrolysis, and enhanced sugar yield, the pretreated biomass was studied by SEM, FT-IR, XRD and H-1 NMR analysis for various structural aberrations that made the biomass more amenable to enzymatic hydrolysis. Insights of underlying functional mechanisms of pretreatment may help in developing new/novel designer pretreatment approaches for proficient biomass conversion. (C) 2020 Elsevier Ltd. All rights reserved.