The excess of oil palm empty fruit bunches (OPEFB) has caused significant ecological issues. Combined peracetic acid (PAA) and ultrasonication were used for OPEFB pretreatment. PAA solutions of varying strengths and duration of ultrasonication at a frequency of 37 kHz at 60°C were examined for OPEFB pretreatment. At 10% PAA for 60 min, the highest levels of cellulose (52.0 ± 0.2%), hemicellulose (39.5 ± 0.2%), and decreased lignin (11.3 ± 0.4%) were achieved. In the ideal pretreatment condition, the highest glucose levels (28 mg/g) and xylose (19 mg/g) was observed. Under a scanning electron microscope (SEM), a significant structural change in OPEFB before and after pretreatment was discovered. FTIR of OPEFB fibre had an absorption peak at 1727 cm^–1, which relates to the hemicellulose’s C=O stretching. From the XRD studies, both crystalline and semi-crystalline zones may be seen in the diffractogram curves of OPEFB cellulose. Neosartorya sp. BCC 18065 produced more xylanase (92 ± 0.03 IU/g) on pretreated OPEFB compared to untreated OPEFB (61 ± 0.01 IU/g). With more xylanase enzymes present during fermentation, xylose production was enhanced. The results demonstrate that structural changes in OPEFB can be changed by PAA-assisted ultrasonication pretreatment, which also improves enzymatic hydrolysis and saccharification