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The Scan Electron Microscope Images (SEM), X-ray Diffraction and Fourier Transform Infrared Spectroscopy (FTIR) dataset has been outlined investigating morphological features change of native sugarcane bagasse, as an agro-industrial lignocellulosic feedstock waste and a potential for cellulose biopolymer extraction, pretreated by alkali (sodium hydroxide) followed by an acid step (sodium bisulfate) in an exothermic in-situ one step, pretreated by acid (sulfuric acid) followed by residual solid fraction alkali pretreatment (sodium hydroxide) in a two separate individual steps and finally after the enzymatic cellulolysis. Data explained herein helps to extend and add to knowledge regarding the impact unlikeness of two different pretreatment methodologies utilize the same chemicals and relatively same concentrations on the cellulosic fiber morphological features and consequently its enzymatic accessibility. This data are related to Egypt Patent Office application, 1349/2017, entitled “In-situ sodium hydroxide-sodium bisulfate sugarcane bagasse pretreatment for biofuel production”,Zohri et al., 2017 [1]. Keywords: Biomass, Pretreatment, In-situ, Cellulose, Enzymatic hydrolysis, Biofuel