The recalcitrant nature of lignocellulosic biomass is attributed to the presence of cellulose, hemicellulose, and lignin. An effective biomass deconstruction method can overcome this challenge and aid in generating fermentable sugars. In order to improve the efficiency of the previously developed assisted single-step acid pretreatment (ASAP) process, the present study introduced a preconditioning (PreC) step prior to ASAP. Among the different preconditioning methods (viz., hot water, steam, steam in the presence of ASAP catalyst, and xylanases) that were tested on rice straw, an increase in delignification (similar to 13%) and reactor solids loading (similar to 50%) were obtained with enzymatic preconditioning. In addition, the characterization of solid and liquid streams arising from all preconditioning and pretreatments revealed the presence of compounds such as polyunsaturated and polysaturated fatty acids as well as organosilanes, which are being reported for the first time in pretreated hydrolysates and solids. Through this study, it is demonstrated that the introduction of a suitable preconditioning method prior to biomass pretreatment can improve bioethanol productivity by similar to 50%.