The glucan-rich fraction, hemicellulosic compounds-rich fraction, and a mixture of both fractions obtained from organosolv pretreatment of oil palm empty fruit bunch (OPEFB) were used as substrates to produce volatile fatty acids (VFAs) in acidogenic fermentation. In this study, the effects of medium adjustment (carbon to nitrogen ratio and trace elements supplementation) and methanogenesis inhibition (through the addition of 2-bromoethanesulfonate or by heat shock) to enhance VFAs yield were investigated. The highest VFA yield was 0.50 ± 0.00 g VFAs/g volatile solid (VS), which was obtained when methanogens were inhibited by heat shock and cultivated in a mixture of glucan-rich and hemicellulosic compounds-rich fractions. Under these conditions, the fermentation produced acetic acid as the only VFA. Based on the results, the mass balance of the whole process (from pretreatment and fermentation) showed the possibility to obtain 30.4 kg acetic acid and 20.3 kg lignin with a 70% purity from 100 kg OPEFB. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.