To convert lignocellulose into transportation fuels and chemical products, pretreatment of the biomass, leading to the separation of the main components (cellulose, hemicellulose, and lignin), is a crucial and still challenging first step. Within this study, the screw press pretreatment of lignocellulosic biomass and its effect on the subsequent chemical pretreatment steps were evaluated. Macroscopic disruption and defibration of reed straw after screw press pretreatment were confirmed by scanning electronic microscopy and dye adsorption studies, showing an enhancement of the accessible surface area. Alkaline and dilute acid hydrolysis was tested to study the release of phenols or sugars from screw-press-treated and non-treated straw materials. To showcase a realistic subsequent chemical pretreatment step, kinetics of enzymatic hydrolysis after a biogenic and biphasic fractionation (OrganoCat) process was tested to evaluate the release of glucose from cut and pressed reed straw. Pretreatment by a screw press enhanced alkaline hydrolysis. A kinetic study showed that screw press pretreatment can reduce processing time and the amount of alkaline chemicals needed. A 75% and a 116% increase in the release of reducing sugars from screw-press-treated wheat and reed straw was detected, respectively. Whereas the OrganoCat fractionation of the screw-press-pretreated reed straw showed no significant improvement, a synergetic effect could be observed in the subsequent enzymatic hydrolysis of the cellulose pulp after combination of both pretreatments in comparison to applying only the cutting mill before chemical fractionation.